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Sample records for thermophilic bacterium caldicellulosiruptor

  1. Complete genome sequences for the anaerobic, extremely thermophilic plant

    Office of Scientific and Technical Information (OSTI)

    biomass-degrading bacteria Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor owensenis, and Caldicellulosiruptor lactoaceticus (Journal Article) | SciTech Connect Complete genome sequences for the anaerobic, extremely thermophilic plant biomass-degrading bacteria Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor owensenis, and

  2. Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus

    DOE Patents [OSTI]

    Tucker, Melvin P.; Grohmann, Karel; Himmel, Michael E.; Mohagheghi, Ali

    1992-01-01

    A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C.sub.1 and C.sub.x types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.

  3. Caldicellulosiruptor Core and Pangenomes Reveal Determinants for

    SciTech Connect (OSTI)

    Blumer-Schuette, Sara E.; Giannone, Richard J; Zurawski, Jeffrey V; Ozdemir, Inci; Ma, Qin; Yin, Yanbin; Xu, Ying; Kataeva, Irena; Poole, Farris; Adams, Michael W. W.; Hamilton-Brehm, Scott; Elkins, James G; Larimer, Frank W; Land, Miriam L; Hauser, Loren John; Cottingham, Robert W; Hettich, Robert {Bob} L; Kelly, Robert M

    2012-01-01

    Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acidpretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.

  4. Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus

    SciTech Connect (OSTI)

    Saw, Jimmy H; Mountain, Bruce W; Feng, Lu; Omelchenko, Marina V; Saito, Jennifer A; Stott, Matthew B; Li, Dan; Zhao, Guang; Wu, Junli; Galperin, Michael Y; Dunfield, Peter F; Wang, Lei; Alam, Maqsudul

    2008-01-01

    Gram-positive bacteria of the genus Anoxybacillus have been found in diverse thermophilic habitats, such as geothermal hot springs and manure, and in processed foods such as gelatin and milk powder. Anoxybacillus flavithermus is a facultatively anaerobic bacterium found in super-saturated silica solutions and in opaline silica sinter. The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life. We report here the complete genome sequence of A. flavithermus strain WK1, isolated from the waste water drain at the Wairakei geothermal power station in New Zealand. It consists of a single chromosome of 2,846,746 base pairs and is predicted to encode 2,863 proteins. In silico genome analysis identified several enzymes that could be involved in silica adaptation and biofilm formation, and their predicted functions were experimentally validated in vitro. Proteomic analysis confirmed the regulation of biofilm-related proteins and crucial enzymes for the synthesis of long-chain polyamines as constituents of silica nanospheres. Microbial fossils preserved in silica and silica sinters are excellent objects for studying ancient life, a new paleobiological frontier. An integrated analysis of the A. flavithermus genome and proteome provides the first glimpse of metabolic adaptation during silicification and sinter formation. Comparative genome analysis suggests an extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli.

  5. Caldicellulosiruptor Core and Pangenomes Reveal Determinants...

    Office of Scientific and Technical Information (OSTI)

    Journal Article: Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Citation Details In-Document Search Title: Caldicellulosiruptor Core and Pangenomes Reveal ...

  6. Fermentation of dilute acid pretreated Populus by Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Yee, Kelsey L.; Rodriguez, Jr., Miguel; Hamilton, Choo Yieng; Hamilton-Brehm, Scott D.; Thompson, Olivia A.; Elkins, James G.; Davison, Brian H.; Mielenz, Jonathan R.

    2015-07-25

    Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three , thermophilic,cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH controlled, anaerobic fermentation conditions, each candidate successfully digested a minimum of 75% of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cellsmore » and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required 120 hours to achieve 75% cellulose utilization. In contrast, the non-cellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required 300 hours to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 grams of total metabolites per gram of loaded glucan, respectively. This data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.« less

  7. Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

    SciTech Connect (OSTI)

    Lochner, Adriane; Giannone, Richard J; Rodriguez, Jr., Miguel; Mielenz, Jonathan R; Keller, Martin; Antranikian, Garabed; Graham, David E; Hettich, Robert {Bob} L

    2011-01-01

    The understanding of microbial cellulose degradation systems is a crucial prerequisite to designing an effective operating process for the bioconversion of lignocellulosic biomass into sustainable biofuels. Relevant in this context are members of the extremely thermophilic Gram-positive bacteria genus Caldicellulosiruptor that have been shown to efficiently degrade cellulose, as well as hemicellulose. Although individual representatives from this genus have been closely examined in bioenergy related studies and single components of their cellulolytic enzyme systems have been described, an overall characterization of the cellulose degradation system is still lacking. To this end, a comparative systems level investigation of two closely related species, Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis, based on label free quantitative proteomics was conducted to determine the protein composition in the organisms secretome over the course of crystalline cellulose fermentations. Mass spectrometric characterizations together with cellulase activity measurements revealed a substantial abundance increase of a few bifunctional multidomain glycosidases that were composed of the domain families 5, 9, 10 and 48, that appear to be important elements for the cellulose degradation process in Caldicellulosiruptor. However, the number and arrangement of these domains varied in the two organisms, and C. bescii enzymes also contained an additional family 44 and 74, indicating significant differences at the species level. Investigation of a glycosidase solution enriched via affinity digestion revealed the presence of highly thermostable enzymes with optimum cellulase activity at 85 C and pH 5 in both organisms. The C. obsidiansis preparation, however, displayed twice the CMCase and Avicelase activity as the C. bescii preparation.

  8. Direct Conversion of Plant Biomass to Ethanol by Engineered Caldicellulosiruptor bescii

    SciTech Connect (OSTI)

    Chung, Daehwan; Cha, Minseok; Guss, Adam M; Westpheling, Janet

    2014-01-01

    Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.

  9. Fermentation of dilute acid pretreated Populus by Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis

    SciTech Connect (OSTI)

    Yee, Kelsey L.; Rodriguez, Jr., Miguel; Hamilton, Choo Yieng; Hamilton-Brehm, Scott D.; Thompson, Olivia A.; Elkins, James G.; Davison, Brian H.; Mielenz, Jonathan R.

    2015-07-25

    Consolidated bioprocessing (CBP), which merges enzyme production, biomass hydrolysis, and fermentation into a single step, has the potential to become an efficient and economic strategy for the bioconversion of lignocellulosic feedstocks to transportation fuels or chemicals. In this study, we evaluated Clostridium thermocellum, Caldicellulosiruptor bescii, and Caldicellulosiruptor obsidiansis, three , thermophilic,cellulolytic, mixed-acid fermenting candidate CBP microorganisms, for their fermentation capabilities using dilute acid pretreated Populus as a model biomass feedstock. Under pH controlled, anaerobic fermentation conditions, each candidate successfully digested a minimum of 75% of the cellulose from dilute acid pretreated Populus, as indicated by an increase in planktonic cells and end-product metabolites and a concurrent decrease in glucan content. C. thermocellum, which employs a cellulosomal approach to biomass degradation, required 120 hours to achieve 75% cellulose utilization. In contrast, the non-cellulosomal, secreted hydrolytic enzyme system of the Caldicellulosiruptor sp. required 300 hours to achieve similar results. End-point fermentation conversions for C. thermocellum, C. bescii, and C. obsidiansis were determined to be 0.29, 0.34, and 0.38 grams of total metabolites per gram of loaded glucan, respectively. This data provide a starting point for future strain engineering efforts that can serve to improve the biomass fermentation capabilities of these three promising candidate CBP platforms.

  10. Thermoflexus hugenholtzii gen. nov., sp. nov., a thermophilic, microaerophilic, filamentous bacterium representing a novel class in the Chloroflexi, Thermoflexia classis nov., and description of Thermoflexaceae fam. nov. and Thermoflexales ord. nov.

    SciTech Connect (OSTI)

    Dodsworth, Jeremy A.; Gevorkian, Jonathan; Despujos, Fairuz; Cole, Jesse; Murugapiran, Senthil K.; Ming, Hong; Li, Wen J.; Zhang, Gengxin; Dohnalkova, Alice; Hedlund, Brian P.

    2014-06-06

    A thermophilic, filamentous, heterotrophic bacterium designated strain JAD2T was isolated from sediment of Great Boiling Spring in Nevada, USA. Cells had an average diameter of 0.3 m and length of 4.0 m, and formed filaments typically ranging in length from 20 m to 200 m. Filaments were negative for the Gram stain reaction, spores were not formed, and motility was not observed. The optimum temperature for growth was 75 C with a range from 67.5-75 C, and the optimum pH for growth was 6.75 with a range from 6.5-7.75. Peptone, tryptone or yeast extract were able to support growth when supplemented with a vitamin solution, but no growth was observed using a variety of defined organic substrates. Strain JAD2T was a facultative microaerophile, with optimal growth at 1% v/v O2 and an upper limit of 8% O2, and anaerobic growth was stimulated by fumarate but inhibited by sulfite and elemental sulfur. The major cellular fatty acids (>5%) were C16:0, C19:0, C18:0, C20:0, and C19:1. The genomic DNA G+C content was 69.3%. Phylogenetic and phylogenomic analyses using 16S rRNA gene sequences and other conserved genes placed JAD2T and other members of the yet-uncultivated GAL35 group within the phylum Chloroflexi, but not within any existing class in this phylum. These results indicate that strain JAD2T is the first cultivated representative of a new lineage within the phylum Chloroflexi, for which we propose the name Thermoflexus hugenholtzii gen. nov., sp. nov., type strain JAD2T, within Thermoflexia classis nov., Thermoflexales ord. nov., and Thermoflexaceae fam. nov.

  11. Thermostable purified endoglucanase from thermophilic bacterium...

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    Biomass and Biofuels Building Energy Efficiency Electricity Transmission Energy Analysis Energy Storage Geothermal Hydrogen and Fuel Cell Hydropower, Wave and Tidal Industrial...

  12. Genome Sequence of Kosmotoga olearia Strain TBF 19.5.1, a Thermophilic Bacterium with a Wide Growth Temperature Range, Isolated from the Troll B Oil Platform in the North Sea

    SciTech Connect (OSTI)

    Swithers, Kristen S; DiPippo, Jonathan L; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Goodwin, Lynne A.; Han, James; Woyke, Tanja; Pitluck, Sam; Pennacchio, Len; Nolan, Matt; Land, Miriam L; Nesbo, Camilla; Gogarten, Peter; Noll, Kenneth M

    2011-01-01

    Kosmotoga olearia strain TBF 19.5.1 is a member of the Thermotogales that grows best at 65 degrees C and very well even at 37 degrees C. Information about this organism is important for understanding the evolution of mesophiles from thermophiles. Its genome sequence reveals extensive gene gains and a large content of mobile genetic elements. It also contains putative hydrogenase genes that have no homologs in the other member of the Thermotogales.

  13. Anaerobic High-Throughput Cultivation Method for Isolation of Thermophiles Using Biomass-Derived Substrates

    SciTech Connect (OSTI)

    Hamilton-Brehm, Scott; Vishnivetskaya, Tatiana A; Allman, Steve L; Mielenz, Jonathan R; Elkins, James G

    2012-01-01

    Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium ther- mocellum (Topt = 55 C), Caldicellulosiruptor obsidiansis (Topt = 78 C) and the fermentative hyperthermo- philes, Pyrococcus furiosus (Topt = 100 C) and Thermotoga maritima (Topt = 80 C). Multi-well plates were incubated at various temperatures for approximately 72 120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD600. Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

  14. Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass

    SciTech Connect (OSTI)

    Young, Jenna; Chung, Daehwan; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2014-10-09

    Background: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii, which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities. Results: A deletion of the celA gene resulted in a dramatic reduction in the microorganism’s ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains. In conclusion: The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.

  15. Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Young, Jenna; Chung, Daehwan; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2014-10-09

    Background: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii,more » which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities. Results: A deletion of the celA gene resulted in a dramatic reduction in the microorganism’s ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains. In conclusion: The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.« less

  16. Anaerobic thermophilic culture

    DOE Patents [OSTI]

    Ljungdahl, Lars G.; Wiegel, Jurgen K. W.

    1981-01-01

    A newly discovered thermophilic anaerobe is described that was isolated in a biologically pure culture and designated Thermoanaerobacter ethanolicus ATCC 3/550. T. Ethanolicus is cultured in aqueous nutrient medium under anaerobic, thermophilic conditions and is used in a novel process for producing ethanol by subjecting carbohydrates, particularly the saccharides, to fermentation action of the new microorganism in a biologically pure culture.

  17. Spatial and temporal dynamics of cellulose degradation and biofilm formation by Caldicellulosiruptor obsidiansis and Clostridium thermocellum Caldicellulosiruptor obsidiansis

    SciTech Connect (OSTI)

    Wang, Zhiwu; Lee, Sueng-Hwan; Elkins, James G; Morrell-Falvey, Jennifer L

    2011-01-01

    Cellulose degradation is one of the major bottlenecks of a consolidated bioprocess that employs cellulolytic bacterial cells as catalysts to produce biofuels from cellulosic biomass. In this study, we investigated the spatial and temporal dynamics of cellulose degradation by Caldicellulosiruptor obsidiansis, which does not produce cellulosomes, and Clostridium thermocellum, which does produce cellulosomes. Results showed that the degradation of either regenerated or natural cellulose was synchronized with biofilm formation, a process characterized by the formation and fusion of numerous crater-like depressions on the cellulose surface. In addition, the dynamics of biofilm formation were similar in both bacteria, regardless of cellulosome production. Only the areas of cellulose surface colonized by microbes were significantly degraded, highlighting the essential role of the cellulolytic biofilm in cellulose utilization. After initial attachment, the microbial biofilm structure remained thin, uniform and dense throughout the experiment. A cellular automaton model, constructed under the assumption that the attached cells divide and produce daughter cells that contribute to the hydrolysis of the adjacent cellulose, can largely simulate the observed process of biofilm formation and cellulose degradation. This study presents a model, based on direct observation, correlating cellulolytic biofilm formation with cellulose degradation.

  18. Anaerobic thermophilic culture system

    DOE Patents [OSTI]

    Ljungdahl, Lars G.; Wiegel, Jurgen K. W.

    1981-01-01

    A mixed culture system of the newly discovered microorganism Thermoanaerobacter ethanolicus ATCC31550 and the microorganism Clostridium thermocellum ATCC31549 is described. In a mixed nutrient culture medium that contains cellulose, these microorganisms have been coupled and cultivated to efficiently ferment cellulose to produce recoverable quantities of ethanol under anaerobic, thermophilic conditions.

  19. Complete Genome Sequence of the Cellulolytic Thermophile Clostridium thermocellum DSM1313

    SciTech Connect (OSTI)

    Feinberg, Lawrence F; Foden, Justine; Barrett, Trisha; Davenport, Karen W.; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Lapidus, Alla L.; Lucas, Susan; Cheng, Jan-Fang; Pitluck, Sam; Woyke, Tanja; Ivanova, N; Mikhailova, Natalia; Land, Miriam L; Hauser, Loren John; Argyros, Aaron; Goodwin, Lynne A.; Hogsett, David; Caiazza, Nicky

    2011-01-01

    Clostridium thermocellum DSM1313 is a thermophilic, anaerobic bacterium with some of the highest rates of cellulose hydrolysis reported. The complete genome sequence reveals a suite of carbohydrate-active enzymes and demonstrates a level of diversity at the species level distinguishing it from the type strain ATCC27405.

  20. Complete genome sequences for the anaerobic, extremely thermophilic...

    Office of Scientific and Technical Information (OSTI)

    Complete genome sequences for the anaerobic, extremely thermophilic plant ... Title: Complete genome sequences for the anaerobic, extremely thermophilic plant ...

  1. Hemicellulases from the ethanologenic thermophile, Thermoanaerobacter ethanolicus and related anaerobic thermophiles. Final report, September 1992--June 1996

    SciTech Connect (OSTI)

    Wiegel, J.

    1998-09-01

    The short term goals of this application were to characterize hemicellulases from anaerobic thermophiles on the biochemical and molecular level to extend the presently limited knowledge of hemicellulases in anaerobic thermophilic bacteria. This objective includes the following tasks: (1) Traditional purification and biochemical/biophysical characterization of xylanases from the newly isolated, slightly alkalitolerant strain NDF190, and the slightly acid-tolerant strain YS485, both with high xylanolytic activities, and of the 4-O-methyl glucuronidase and arabinosidase from strain NDF190 and the acetyl (xylan) esterase from T. ethanolicus. This also includes determining the N-terminal sequences and obtaining gene probes. (2) Elucidation of the regulation of hemicellulolytic enzymes in anaerobic thermophiles. (3) To clone into E. coli and identify the multiplicity of the enzymes involved in hemicellulose degradation by T. ethanolicus and other suitable organisms. (4) To purify and characterize the recombinant enzymes with the goal of identifying the best enzymes for cloning into the ethanologenic T. ethanolicus to obtain an optimized hemicellulose utilization by this bacterium.

  2. Isolation of butyrate-utilizing bacteria from thermophilic and mesophilic methane-producing ecosystems

    SciTech Connect (OSTI)

    Henson, J.M.

    1983-01-01

    The ability of various ecosystems to convert butyrate to methane was studied in order to isolate the bacteria responsible for the conversion. When thermophilic digester sludge was enriched with butyrate, methane was produced without a lag period. Marine sediments enriched with butyrate required a 2-week incubation period before methanogenesis began. A thermophilic digester was studied in more detail and found by most-probable-number enumeration to have ca. 5 x 10/sup 6/ butyrate-utilizing bactera/ml of sludge. A thermophilic butyrate-utilizing bacterium was isolated in coculture with Methanobacterium thermoautotrophicum and a Methanosarcina sp. This bacterium was a gram-negative, slightly curved rod that occurred singly, was nonmotile, and did not appear to produce spores. The thermophilic digester was infused with butyrate at the rate of 10 ..mu..moles/ml of sludge per day. Biogas production increased by 150%, with the percentage of methane increasing from 58% to 68%. Acetate, propionate, and butyrate did not accumulate. Butyrate-utilizing enrichments from mesophilic ecosystems were used in obtaining cocultures of butyrate-utilizing bacteria. These cocultures served as inocula for attempts to isolate pure cultures of butyrate-utilizing bacteria by use of hydrogenase-containing membrane fragments of Escherichia coli. After a 3-week incubation period, colonies appeared only in inoculated tubes that contained membrane fragments and butyrate.

  3. Copy of Synthetic Biology of Novel Thermophilic Bacteria for...

    Office of Scientific and Technical Information (OSTI)

    Copy of Synthetic Biology of Novel Thermophilic Bacteria for Enhanced Production of ... Citation Details In-Document Search Title: Copy of Synthetic Biology of Novel Thermophilic ...

  4. Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Groom, Joseph; Chung, Daehwan; Olson, Daniel G.; Lynd, Lee R.; Guss, Adam M.; Westpheling, Janet

    2016-01-29

    Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproduciblymore » transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.« less

  5. Thermophilic microbes in ethanol production

    SciTech Connect (OSTI)

    Slapack, G.E.; Russell, I.; Stewart, G.G.

    1987-01-01

    General and specific properties of thermophilic ethanol-producing bacteria are reviewed and their relative merits in ethanol production assessed. The studies examine the use of bacteria in mono- and co-culture fermentations for ethanol production from cellulosics; in particular, the cellulase system of Clostridium thermocellum is considered. Thermotolerant yeasts and physiological factors influencing their growth and fermentation at high temperatures are discussed. Emphasis is placed on multidisciplinary approaches to develop economical processes for ethanol production at high temperatures. Relevant topics considered include: adaptation, nutrition, heat shock, ethanol tolerance, metabolic control, genetic improvement, and fermentation/process design. General aspects of thermophily for both bacteria and yeasts (definitions, ecological aspects, merits and limitations, other industrial uses, thermostability of cellular components, and consequences of thermophilic fermentation) are discussed and the volume references over 1100 relevant articles.

  6. Consolidated bioprocessing method using thermophilic microorganisms

    DOE Patents [OSTI]

    Mielenz, Jonathan Richard

    2016-02-02

    The present invention is directed to a method of converting biomass to biofuel, and particularly to a consolidated bioprocessing method using a co-culture of thermophilic and extremely thermophilic microorganisms which collectively can ferment the hexose and pentose sugars produced by degradation of cellulose and hemicelluloses at high substrate conversion rates. A culture medium therefor is also provided as well as use of the methods to produce and recover cellulosic ethanol.

  7. Nitrogen and Sulfur Requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on Cellulosic Substrates in Minimal Nutrient Media

    SciTech Connect (OSTI)

    Kridelbaugh, Donna M; Nelson, Josh C; Engle, Nancy L; Tschaplinski, Timothy J; Graham, David E

    2013-01-01

    Growth media for cellulolytic Clostridium thermocellum and Caldicellulosiruptor bescii bacteria usually contain excess nutrients that would increase costs for consolidated bioprocessing for biofuel production and create a waste stream with nitrogen, sulfur and phosphate. C. thermocellum was grown on crystalline cellulose with varying concentrations of nitrogen and sulfur compounds, and growth rate and alcohol production response curves were determined. Both bacteria assimilated sulfate in the presence of ascorbate reductant, increasing the ratio of oxidized to reduced fermentation products. From these results, a low ionic strength, defined minimal nutrient medium with decreased nitrogen, sulfur, phosphate and vitamin supplements was developed for the fermentation of cellobiose, cellulose and acid-pretreated Populus. Carbon and electron balance calculations indicate the unidentified residual fermentation products must include highly reduced molecules. Both bacterial populations were maintained in co-cultures with substrates containing xylan or hemicellulose in defined medium with sulfate and basal vitamin supplements.

  8. Degradation of xylan by a new strain of thermophilic Clostridium

    SciTech Connect (OSTI)

    Boyce, E.N.

    1988-01-01

    The intent of the research has been the isolation of a thermophilic, polysaccharide-degrading anaerobe which could prove suitable for coculturing with organisms such as Clostridium thermocellum, a prominent cellulose-degrading bacterium. The author has isolated such an organism from Kansas soil. The isolate virgorously degrades xylan, a hemicellulose, as well as several starchy substrates and other polysaccharides, though not cellulose. In addition, the isolate ferments all common mono- and di-saccharide components of plant polysaccharides. Though its fermentation is largely acidic, it also produces significant amounts of enthanol and n-butanol. Biochemical and metabolic characterization of the isolate have allowed it to be distinguished from previously-reported strains of the genus Clostridium, though currently insufficient evidence is available to report it as a new species. Initial studies of the isolate's xylan-degrading system reveal that the organism produces at least six separate xylanases when the isolate grows in media containing xylose, a component of xylan. In xylan medium, the isolate also produces a yellow, high-charged substance which co-migrates electrophoretically with its active xylanase(s). This substance may be analogous to the yellow substrate affinity substance (YAS) produced by C. thermocellum in cellulose medium.

  9. Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Chung, Daehwan; Young, Jenna; Cha, Minseok; Brunecky, Roman; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2015-08-13

    The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5more » domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.« less

  10. The hemicellulases from the ethanologenic thermophile, Thermoanaerobacter ethanolicus and similar anaerobic thermophiles. Annual technical progress report

    SciTech Connect (OSTI)

    Wiegel, J.

    1995-07-01

    A Xylanase was fractionated from Thermoanaerobacter ethanolicus, an ethanologenic thermophile, and the preparation so obtained was used to determined enzymatic parameters such as pH profile of enzyme activity. The ability of various mono- and di-saccharides as well as temperature variations to induce this enzyme activity were studied.

  11. Copy of Synthetic Biology of Novel Thermophilic Bacteria for Enhanced

    Office of Scientific and Technical Information (OSTI)

    Production of Ethanol from 5-Carbon Sugars (LDRD %23 105944). (Conference) | SciTech Connect Copy of Synthetic Biology of Novel Thermophilic Bacteria for Enhanced Production of Ethanol from 5-Carbon Sugars (LDRD %23 105944). Citation Details In-Document Search Title: Copy of Synthetic Biology of Novel Thermophilic Bacteria for Enhanced Production of Ethanol from 5-Carbon Sugars (LDRD %23 105944). Abstract not provided. Authors: Reichmuth, David ; Kozina, Carol L. ; Sale, Kenneth L. ;

  12. Synthetic Biology of Novel Thermophilic Bacteria for Enhanced Production of

    Office of Scientific and Technical Information (OSTI)

    Ethanol from 5-Carbon Sugars (LDRD %23 105944). (Conference) | SciTech Connect Synthetic Biology of Novel Thermophilic Bacteria for Enhanced Production of Ethanol from 5-Carbon Sugars (LDRD %23 105944). Citation Details In-Document Search Title: Synthetic Biology of Novel Thermophilic Bacteria for Enhanced Production of Ethanol from 5-Carbon Sugars (LDRD %23 105944). Abstract not provided. Authors: Sapra, Rajat ; Reichmuth, David ; Kozina, Carol L. ; Sale, Kenneth L. ; Keasling, Jay ; Tang,

  13. Thermophilic lignocellulose deconstruction (Journal Article) | SciTech

    Office of Scientific and Technical Information (OSTI)

    Connect Thermophilic lignocellulose deconstruction Citation Details In-Document Search Title: Thermophilic lignocellulose deconstruction Authors: Blumer-Schuette, Sara E. [1] ; Brown, Steven D [2] ; Sander, Kyle B [2] ; Bayer, Edward A [2] ; Kataeva, Irena [3] ; Zurawski, Jeffrey V [1] ; Conway, Jonathan M [1] ; Adams, Michael W. W. [3] ; Kelly, Robert M [1] + Show Author Affiliations North Carolina State University [North Carolina State University ORNL [ORNL University of Georgia, Athens,

  14. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    SciTech Connect (OSTI)

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.

  15. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  16. Production of ethanol from lignocellulosic materials using thermophilic bacteria

    SciTech Connect (OSTI)

    Lynd, L.R.

    1987-01-01

    The production of ethanol from lignocellulosic materials, e.g. wood, agricultural residues, and municipal solid wastes, is considered. The conversion of these materials to ethanol in the US could annually yield approximately 430 million tons ethanol, or about 9.8 quads, within the next 20 years. Thermophilic bacteria have advantages over yeasts for ethanol production because various species produce an active cellulase enzyme and utilize pentose sugars. However thermophiles have lower ethanol tolerance and usually lower ethanol yields. The potential of thermophilic ethanol production from hardwood chips is examined in detail. It is concluded that if high ethanol yield can be achieved this process could have economics competitive with either ethanol production from corn via yeast or synthetic production from ethylene. Low ethanol tolerance is not a major problem provided concentrations {ge} 1.5% are produced, ethanol is continuously removed from the fermentor, and IHOSR/extractive distillation is employed. Research was undertaken aimed at closing the gap between the attractive potential of thermophiles for ethanol production, and that which is possible based on present knowledge, which is not practical. Major topics were the activity of Clostridium thermocellum cellulase on pretreated mixed hardwood and Avicel in vivo, continuous culture of C. thermocellum on pretreated mixed hardwood and Avicel, and the continuous culture of Clostridium thermosaccharolyticum at high xylose concentrations in the presence and absence of ethanol removal.

  17. Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

    SciTech Connect (OSTI)

    Chung, Daehwan; Young, Jenna; Cha, Minseok; Brunecky, Roman; Bomble, Yannick J.; Himmel, Michael E.; Westpheling, Janet

    2015-08-13

    The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5 domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.

  18. Ultrafast time-resolved spectroscopy of the light-harvesting complex 2 (LH2) from the photosynthetic bacterium Thermochromatium tepidum

    SciTech Connect (OSTI)

    Niedzwiedzki, Dariusz M.; Fuciman, Marcel; Kobayashi, Masayuki; Frank, Harry A.; Blankenship, Robert E.

    2011-10-08

    The light-harvesting complex 2 from the thermophilic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption and fluorescence, sub-nanosecond-time-resolved fluorescence and femtosecond time-resolved transient absorption spectroscopy. The measurements were performed at room temperature and at 10 K. The combination of both ultrafast and steady-state optical spectroscopy methods at ambient and cryogenic temperatures allowed the detailed study of carotenoid (Car)-to-bacteriochlorophyll (BChl) as well BChl-to-BChl excitation energy transfer in the complex. The studies show that the dominant Cars rhodopin (N = 11) and spirilloxanthin (N = 13) do not play a significant role as supportive energy donors for BChl a. This is related with their photophysical properties regulated by long ?-electron conjugation. On the other hand, such properties favor some of the Cars, particularly spirilloxanthin (N = 13) to play the role of the direct quencher of the excited singlet state of BChl.

  19. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

    SciTech Connect (OSTI)

    Berka, Randy M.; Grigoriev, Igor V.; Otillar, Robert; Salamov, Asaf; Grimwood, Jane; Reid, Ian; Ishmael, Nadeeza; John, Tricia; Darmond, Corinne; Moisan, Marie-Claude; Henrissat, Bernard; Coutinho, Pedro M.; Lombard, Vincent; Natvig, Donald O.; Lindquist, Erika; Schmutz, Jeremy; Lucas, Susan; Harris, Paul; Powlowski, Justin; Bellemare, Annie; Taylor, David; Butler, Gregory; de Vries, Ronald P.; Allijn, Iris E.; van den Brink, Joost; Ushinsky, Sophia; Storms, Reginald; Powell, Amy J.; Paulsen, Ian T.; Elbourne, Liam D. H.; Baker, Scott. E.; Magnuson, Jon; LaBoissiere, Sylvie; Clutterbuck, A. John; Martinez, Diego; Wogulis, Mark; Lopez de Leon, Alfredo; Rey, Michael W.; Tsang, Adrian

    2011-05-16

    Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

  20. Pathway engineering and organism development for ethanol production from cellulosic biomass using thermophilic bacteria

    SciTech Connect (OSTI)

    Hogsett, D.A.L.; Klapatch, T.A.; Lynd, L.R.

    1995-12-01

    Thermophilic bacteria collectively exemplify organisms that produce both cellulose and ethanol while fermenting both the cellulose and hemicellulose components of biomass. As a result, thermophiles could be the basis for highly streamlined and cost-effective processes for production of renewable fuels and chemicals. Recent research results involving ethanol production from thermophilic bacteria will be presented, with a primary focus on work pursuant to molecularly-based pathway engineering to increase ethanol selectivity. Specifically, we will describe the restriction endonuclease systems operative in Clostridium thermocellum and C. thermosaccharolyticum, as well as efforts to document and improve transformation of these organisms and to clone key catabolic enzymes. In addition, selected results from fermentation studies will be presented as necessary in order to present a perspective on the status of thermophilic ethanol production.

  1. Biocorrosive Thermophilic Microbial Communities in Alaskan North Slope Oil Facilities

    SciTech Connect (OSTI)

    Duncan, Kathleen E.; Gieg, Lisa M.; Parisi, Victoria A.; Tanner, Ralph S.; Green Tringe, Susannah; Bristow, Jim; Suflita, Joseph M.

    2009-09-16

    Corrosion of metallic oilfield pipelines by microorganisms is a costly but poorly understood phenomenon, with standard treatment methods targeting mesophilic sulfatereducing bacteria. In assessing biocorrosion potential at an Alaskan North Slope oil field, we identified thermophilic hydrogen-using methanogens, syntrophic bacteria, peptideand amino acid-fermenting bacteria, iron reducers, sulfur/thiosulfate-reducing bacteria and sulfate-reducing archaea. These microbes can stimulate metal corrosion through production of organic acids, CO2, sulfur species, and via hydrogen oxidation and iron reduction, implicating many more types of organisms than are currently targeted. Micromolar quantities of putative anaerobic metabolites of C1-C4 n-alkanes in pipeline fluids were detected, implying that these low molecular weight hydrocarbons, routinely injected into reservoirs for oil recovery purposes, are biodegraded and provide biocorrosive microbial communities with an important source of nutrients.

  2. Pathway engineering to improve ethanol production by thermophilic bacteria

    SciTech Connect (OSTI)

    Lynd, L.R.

    1998-12-31

    Continuation of a research project jointly funded by the NSF and DOE is proposed. The primary project goal is to develop and characterize strains of C. thermocellum and C. thermosaccharolyticum having ethanol selectivity similar to more convenient ethanol-producing organisms. An additional goal is to document the maximum concentration of ethanol that can be produced by thermophiles. These goals build on results from the previous project, including development of most of the genetic tools required for pathway engineering in the target organisms. As well, we demonstrated that the tolerance of C. thermosaccharolyticum to added ethanol is sufficiently high to allow practical utilization should similar tolerance to produced ethanol be demonstrated, and that inhibition by neutralizing agents may explain the limited concentrations of ethanol produced in studies to date. Task 1 involves optimization of electrotransformation, using either modified conditions or alternative plasmids to improve upon the low but reproducible transformation, frequencies we have obtained thus far.

  3. Comparing residue clusters from thermophilic and mesophilic enzymes reveals adaptive mechanisms

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Sammond, Deanne W.; Kastelowitz, Noah; Himmel, Michael E.; Yin, Hang; Crowley, Michael F.; Bomble, Yannick J.

    2016-01-07

    Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research.more » Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. As a result, the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.« less

  4. Community dynamics and glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass

    SciTech Connect (OSTI)

    Gladden, J.M.; Allgaier, M.; Miller, C.S.; Hazen, T.C.; VanderGheynst, J.S.; Hugenholtz, P.; Simmons, B.A.; Singer, S.W.

    2011-05-01

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60 C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80 C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  5. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    SciTech Connect (OSTI)

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  6. The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

    SciTech Connect (OSTI)

    Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin; Sacchettini, James C. (TAM)

    2010-07-20

    The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.

  7. High-solids enrichment of thermophilic microbial communities and their enzymes on bioenergy feedstocks

    SciTech Connect (OSTI)

    Reddy, A. P.; Allgaier, M.; Singer, S.W.; Hazen, T.C.; Simmons, B.A.; Hugenholtz, P.; VanderGheynst, J.S.

    2011-04-01

    Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switchgrass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment.

  8. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    SciTech Connect (OSTI)

    Xie, Gary; Detter, John C; Bruce, David C; Challacombe, Jean F; Brettin, Thomas S; Necsulea, Anamaria; Daubin, Vincent; Medigue, Claudine; Adney, William S; Xu, Xin C; Lapidus, Alla; Pujic, Pierre; Berry, Alison M; Barabote, Ravi D; Leu, David; Normand, Phillipe

    2009-01-01

    We present here the complete 2.4 MB genome of the actinobacterial thermophile, Acidothermus cellulolyticus 11B, that surprisingly reveals thermophilic amino acid usage in only the cytosolic subproteome rather than its whole proteome. Thermophilic amino acid usage in the partial proteome implies a recent, ongoing evolution of the A. cellulolyticus genome since its divergence about 200-250 million years ago from its closest phylogenetic neighbor Frankia, a mesophilic plant symbiont. Differential amino acid usage in the predicted subproteomes of A. cellulolyticus likely reflects a stepwise evolutionary process of modern thermophiles in general. An unusual occurrence of higher G+C in the non-coding DNA than in the transcribed genome reinforces a late evolution from a higher G+C common ancestor. Comparative analyses of the A. cellulolyticus genome with those of Frankia and other closely-related actinobacteria revealed that A. cellulolyticus genes exhibit reciprocal purine preferences at the first and third codon positions, perhaps reflecting a subtle preference for the dinucleotide AG in its mRNAs, a possible adaptation to a thermophilic environment. Other interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote reveal streamlining for adaptation to its specialized ecological niche. These include a low occurrence of pseudo genes or mobile genetic elements, a flagellar gene complement previously unknown in this organism, and presence of laterally-acquired genomic islands of likely ecophysiological value. New glycoside hydrolases relevant for lignocellulosic biomass deconstruction were identified in the genome, indicating a diverse biomass-degrading enzyme repertoire several-fold greater than previously characterized, and significantly elevating the industrial value of this organism.

  9. Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations

    SciTech Connect (OSTI)

    Xie, Gary; Detter, Chris; Bruce, David; Challacome, Jean F; Brettin, Thomas S; Barabote, Ravi D; Leu, David; Normand, Philippe; Necsula, Anamaria; Daubin, Vincent; Medigue, Claudine; Xu, Xin C; Lapidus, Alla; Pujic, Pierre; Richardson, Paul; Berry, Alison M

    2008-01-01

    We present here the complete 2.4 MB genome of the actinobacterial thermophile, Acidothermus cellulolyticus lIB, that surprisingly reveals thermophilic amino acid usage in only the cytosolic subproteome rather than its whole proteome. Thermophilic amino acid usage in the partial proteome implies a recent, ongoing evolution of the A. cellulolyticus genome since its divergence about 200-250 million years ago from its closest phylogenetic neighbor Frankia, a mesophilic plant symbiont. Differential amino acid usage in the predicted subproteomes of A. cellulolyticus likely reflects a stepwise evolutionary process of modern thermophiles in general. An unusual occurrence of higher G+C in the non-coding DNA than in the transcribed genome reinforces a late evolution from a higher G+C common ancestor. Comparative analyses of the A. cellulolyticus genome with those of Frankia and other closely-related actinobacteria revealed that A. cellulolyticus genes exhibit reciprocal purine preferences at the first and third codon positions, perhaps reflecting a subtle preference for the dinucleotide AG in its mRNAs, a possible adaptation to a thermophilic environment. Other interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote reveal streamlining for adaptation to its specialized ecological niche. These include a low occurrence of pseudogenes or mobile genetic elements, a flagellar gene complement previously unknown in this organism, and presence of laterally-acquired genomic islands of likely ecophysiological value. New glycoside hydrolases relevant for lignocellulosic biomass deconstruction were identified in the genome, indicating a diverse biomass-degrading enzyme repertoire several-fold greater than previously characterized, and significantly elevating the industrial value of this organism.

  10. Near-complete genome sequence of the cellulolytic Bacterium Bacteroides (Pseudobacteroides) cellulosolvens ATCC 35603

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Dassa, Bareket; Utturkar, Sagar M.; Hurt, Richard A.; Klingeman, Dawn Marie; Keller, Martin; Xu, Jian; Reddy, Harish Kumar; Borovok, Ilya; Grinberg, Inna Rozman; Lamed, Raphael; et al

    2015-09-24

    We report the single-contig genome sequence of the anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens. The bacterium produces a particularly elaborate cellulosome system, whereas the types of cohesin-dockerin interactions are opposite of other known cellulosome systems: cell-surface attachment is thus mediated via type-I interactions whereas enzymes are integrated via type-II interactions.

  11. Construction of a bacterium to convert cellulose to ethanol. Final report

    SciTech Connect (OSTI)

    Bellamy, W.D.

    1984-03-01

    In the strains of thermophilic actinomycetes examined, cellobiase (CBase) and Beta-glucosidase (BGSase) were determined to be separate enzymes. Both enzymes are induced by cellulose, cellobiose and lactose. A number of strains do not utilize lactose. Lactose does not induce endocellulase (CMCase) in any of the strains examined. In all the strains examined, the CBase and BGSase were far more heat labile than the extracellular CMCase. The 50% survival time at 60/sup 0/C is as follows: CMCase, 24 hrs; CBase, 10 to 11 hrs; BGSase, 2 to 5 hrs. The BGSase and CBase of Clostridium thermocellum are more heat resistant with 50% survival times: BGSase, 14 hrs; CBase, 41 hrs. Whey permeate is an adequate substrate for a number of strains if supplemented with 0.1% yeast extract or biotin and thiamine. It is speculated that whey permeate could be used for commercial production of CBase and BGSase. All attempts to produce a thermophilic bacillus that was ethanol-tolerant and produced high yields of ethanol by induced mutation using ultraviolet radiation and N-methyl-N'-nitrosogunidine as mutagens were unsuccessful. No evidence was observed that the Acetyl-S-CoA metabolic pathway was deleted or suppressed. Some of the mutants appeared to have decreased yields of lactic acid. A satisfactory screening procedure for selection of high ethanol producing colonies was not found. The screening for low acid production was tedious and time consuming. Because of the failure to find or produce a thermophile with high yields of ethanol, and because all previous work as reported in the literature also yielded poor results, it may be impossible to produce an ethanol-tolerant high yielding thermophilic microorganism. The essential proteins may be unstable at greater than 7% ethanol at 55 to 66/sup 0/C. 48 references, 6 figures, 16 tables.

  12. Bioconversion of methane to lactate by an obligate methanotrophic bacterium

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Henard, Calvin A.; Smith, Holly; Dowe, Nancy; Kalyuzhnaya, Marina G.; Pienkos, Philip T.; Guarnieri, Michael T.

    2016-02-23

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resultedmore » in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels.« less

  13. High rate mesophilic, thermophilic, and temperature phased anaerobic digestion of waste activated sludge: A pilot scale study

    SciTech Connect (OSTI)

    Bolzonella, David; Cavinato, Cristina; Fatone, Francesco; Pavan, Paolo; Cecchi, Franco

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer High temperatures were tested in single and two-stage anaerobic digestion of waste activated sludge. Black-Right-Pointing-Pointer The increased temperature demonstrated the possibility of improving typical yields of the conventional mesophilic process. Black-Right-Pointing-Pointer The temperature phased anaerobic digestion process (65 + 55 Degree-Sign C) showed the best performances with yields of 0.49 m{sup 3}/kgVS{sub fed}. Black-Right-Pointing-Pointer Ammonia and phosphate released from solids destruction determined the precipitation of struvite in the reactor. - Abstract: The paper reports the findings of a two-year pilot scale experimental trial for the mesophilic (35 Degree-Sign C), thermophilic (55 Degree-Sign C) and temperature phased (65 + 55 Degree-Sign C) anaerobic digestion of waste activated sludge. During the mesophilic and thermophilic runs, the reactor operated at an organic loading rate of 2.2 kgVS/m{sup 3}d and a hydraulic retention time of 20 days. In the temperature phased run, the first reactor operated at an organic loading rate of 15 kgVS/m{sup 3}d and a hydraulic retention time of 2 days while the second reactor operated at an organic loading rate of 2.2 kgVS/m{sup 3}d and a hydraulic retention time of 18 days (20 days for the whole temperature phased system). The performance of the reactor improved with increases in temperature. The COD removal increased from 35% in mesophilic conditions, to 45% in thermophilic conditions, and 55% in the two stage temperature phased system. As a consequence, the specific biogas production increased from 0.33 to 0.45 and to 0.49 m{sup 3}/kgVS{sub fed} at 35, 55, and 65 + 55 Degree-Sign C, respectively. The extreme thermophilic reactor working at 65 Degree-Sign C showed a high hydrolytic capability and a specific yield of 0.33 gCOD (soluble) per gVS{sub fed}. The effluent of the extreme thermophilic reactor showed an average concentration of soluble COD and volatile fatty acids of 20 and 9 g/l, respectively. Acetic and propionic acids were the main compounds found in the acids mixture. Because of the improved digestion efficiency, organic nitrogen and phosphorus were solubilised in the bulk. Their concentration, however, did not increase as expected because of the formation of salts of hydroxyapatite and struvite inside the reactor.

  14. Toxicity of Select Organic Acids to the Slightly Thermophilic Acidophile Acidithiobaccillus Caldus

    SciTech Connect (OSTI)

    John E Aston; William A Apel; Brady D Lee; Brent M Peyton

    2009-02-01

    Acidithiobacillus caldus is a thermophilic acidophile found in commercial biomining, acid mine drainage systems, and natural environments. Previous work has characterized A. caldus as a chemolithotrophic autotroph capable of utilizing reduced sulfur compounds under aerobic conditions. Organic acids are especially toxic to chemolithotrophs in low-pH environments, where they diffuse more readily into the cell and deprotonate within the cytoplasm. In the present study, the toxic effects of oxaloacetate, pyruvate, 2-ketoglutarate, acetate, malate, succinate, and fumarate on A. caldus strain BC13 were examined under batch conditions. All tested organic acids exhibited some inhibitory effect. Oxaloacetate was observed to inhibit growth completely at a concentration of 250 M, whereas other organic acids were completely inhibitory at concentrations of between 1,000 and 5,000 M. In these experiments, the measured concentrations of organic acids decreased with time, indicating uptake or assimilation by the cells. Phospholipid fatty acid analyses indicated an effect of organic acids on the cellular envelope. Notable differences included an increase in cyclic fatty acids in the presence of organic acids, indicating possible instability of the cellular envelope. This was supported by field emission scanning-electron micrographs showing blebbing and sluffing in cells grown in the presence of organic acids.

  15. Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic ThermophileAlvinella pompejana

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Kashiwagi, Sayo; Kuraoka, Isao; Fujiwara, Yoshie; Hitomi, Kenichi; Cheng, Quen J.; Fuss, Jill O.; Shin, David S.; Masutani, Chikahide; Tainer, John A.; Hanaoka, Fumio; et al

    2010-01-01

    Human DNA polymerase?(HsPol?) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-inducedcis-syncyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPol?from the thermophilic wormAlvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPol?shares sequence homology with HsPol?and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrateAlvinella'senvironment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPol?is more thermostable than HsPol?, as expected from its habitat temperature.moreMoreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPol?provides a robust, human-like Pol?that is more active after exposure to high temperatures and organic solvents.less

  16. Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Kashiwagi, Sayo; Kuraoka, Isao; Fujiwara, Yoshie; Hitomi, Kenichi; Cheng, Quen J.; Fuss, Jill O.; Shin, David S.; Masutani, Chikahide; Tainer, John A.; Hanaoka, Fumio; et al

    2010-01-01

    Humore » man DNA polymerase η (HsPol η ) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPol η from the thermophilic worm Alvinella pompejana , which inhabits deep-sea hydrothermal vent chimneys. ApPol η shares sequence homology with HsPol η and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPol η is more thermostable than HsPol η , as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPol η provides a robust, human-like Pol η that is more active after exposure to high temperatures and organic solvents.« less

  17. Isolation of cellulolytic anaerobic extreme thermophiles from New Zealand thermal sites

    SciTech Connect (OSTI)

    Sissons, C.H.; Sharrock, K.R.; Daniel, R.M.; Morgan, H.W.

    1987-04-01

    Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75/sup 0/C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75/sup 0/C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75/sup 0/C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases had lower rates of free endocellulase accumulation at 75/sup 0/C than did Clostridium thermocellum at 60/sup 0/C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85/sup 0/C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.

  18. Differences in xylan degradation by various noncellulolytic thermophilic anaerobes and Clostridium thermocellum

    SciTech Connect (OSTI)

    Wiegel, J.; Mothershed, C.P.; Puls, J.

    1985-03-01

    Hemicellulose fractions with a predetermined distribution of xylose, xylooligomers, and xylan fractions were obtained through steam explosion of wood by the steam explosion-extraction process. A differential utilization of various molecular-weight fractions by several thermophilic anaerobic bacteria was determined during their growth on the hemicellulose preparations. Clostridium thermocellum (60/sup 0/C) first utilized the high-molecular-weight fractions (polymerization degree of 15 to 40 xylose units). Xylose and xylooligomers of n = 2 to 5 accumulated while C. thermocellum was not growing, as evident from the fermentation products formed. Whereas the xylan was hydrolyzed and the small oligoxylans were utilized after more than 100 h of incubation, xylose was not significantly utilized. In contrast to this, C. thermohydrosulfuricum (70/sup 8/C) and Thermoanaerobium brockii (70/sup 8/C) utilized xylose first and then xylooligomers of n = 2 to 5, but xylooligomers of n greater than 6 were only slowly utilized. Thermoanaerobacter ethanolicus (70/sup 0/C), Thermobacteroids acetoethylicus (70/sup 0/C), and C. thermosaccharolyticum (60/sup 0/) utilized xylose preferentially. Xylooligomers of n = 2 to 5 and n = 6 and greater were apparently concomitantly utilized without significant differences. In contrast to C. thermocellum, the non-cellulolytic organisms grew during xylan hydrolysis, producing ethanol, lactate, acetate, CO/sub 2/, and H/sub 2/.

  19. Complete genome of the cellyloytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evloutionary adaptations

    SciTech Connect (OSTI)

    Barabote, Ravi D.; Xie, Gary; Leu, David H.; Normand, Philippe; Necsulea, Anamaria; Daubin, Vincent; Medigue, Claudine; Adney, William S.; Xu,Xin Clare; Lapidus, Alla; Detter, Chris; Pujic, Petar; Bruce, David; Lavire, Celine; Challacombe, Jean F.; Brettin, Thomas S.; Berry, Alison M.

    2009-01-01

    We present here the complete 2.4 Mb genome of the cellulolytic actinobacterial thermophile, Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized, and significantly elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. A novel feature of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymes is that they are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. Interestingly, CBM3 was found to be always N-terminal to CBM2, suggesting a functional constraint driving this organization. While the catalytic domains of these modular enzymes are either diverse or unrelated, the CBMs were found to be highly conserved in sequence and may suggest selective substrate-binding interactions. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and non-coding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles, and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote include a low occurrence of pseudogenes or mobile genetic elements, an unexpected complement of flagellar genes, and presence of three laterally-acquired genomic islands of likely ecophysiological value.

  20. Ligand-induced conformational changes in a thermophilic ribose-binding protein

    SciTech Connect (OSTI)

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2009-05-21

    Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (appTm value is 108 C) than the mesophilic Escherichia coli homolog (ecRBP) (appTm value is 56 C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.

  1. 1H, 13C, and 15N backbone and side chain resonance assignments of thermophilic Geobacillus kaustophilus cyclophilin-A

    SciTech Connect (OSTI)

    Holliday, Michael; Zhang, Fengli; Isern, Nancy G.; Armstrong, Geoffrey S.; Eisenmesser, Elan Z.

    2014-04-01

    Cyclophilins catalyze the reversible peptidyl-prolyl isomerization of their substrates and are present across all kingdoms of life from humans to bacteria. Although numerous biological roles have now been discovered for cyclophilins, their function was initially ascribed to their chaperone-like activity in protein folding where they catalyze the often rate-limiting step of proline isomerization. This chaperone-like activity may be especially important under extreme conditions where cyclophilins are often over expressed, such as in tumors for human cyclophilins {Lee, 2010 #1167}, but also in organisms that thrive under extreme conditions, such as theromophilic bacteria. Moreover, the reversible nature of the peptidyl-prolyl isomerization reaction catalyzed by cyclophilins has allowed these enzymes to serve as model systems for probing the role of conformational changes during catalytic turnover {Eisenmesser, 2002 #20;Eisenmesser, 2005 #203}. Thus, we present here the resonance assignments of a thermophilic cyclophilin from Geobacillus kaustophilus derived from deep-sea sediment {Takami, 2004 #1384}. This thermophilic cyclophilin may now be studied at a variety of temperatures to provide insight into the comparative structure, dynamics, and catalytic mechanism of cyclophilins.

  2. In Situ Expression of Acidic and Thermophilic Carbohydrate Active Enzymes by Filamentous Fungi (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    SciTech Connect (OSTI)

    Mosier, Annika [Stanford University] [Stanford University

    2012-03-22

    Annika Mosier, graduate student from Stanford University presents a talk titled "In Situ Expression of Acidic and Thermophilic Carbohydrate Active Enzymes by Filamentous Fungi" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 22, 2012 in Walnut Creek, Calif

  3. In Situ Expression of Acidic and Thermophilic Carbohydrate Active Enzymes by Filamentous Fungi (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    ScienceCinema (OSTI)

    Mosier, Annika [Stanford University

    2013-01-22

    Annika Mosier, graduate student from Stanford University presents a talk titled "In Situ Expression of Acidic and Thermophilic Carbohydrate Active Enzymes by Filamentous Fungi" at the JGI User 7th Annual Genomics of Energy & Environment Meeting on March 22, 2012 in Walnut Creek, Calif

  4. Dry-thermophilic anaerobic digestion of organic fraction of municipal solid waste: Methane production modeling

    SciTech Connect (OSTI)

    Fdez-Gueelfo, L.A.; Alvarez-Gallego, C.; Sales, D.; Romero Garcia, L.I.

    2012-03-15

    Highlights: Black-Right-Pointing-Pointer Methane generation may be modeled by means of modified product generation model of Romero Garcia (1991). Black-Right-Pointing-Pointer Organic matter content and particle size influence the kinetic parameters. Black-Right-Pointing-Pointer Higher organic matter content and lower particle size enhance the biomethanization. - Abstract: The influence of particle size and organic matter content of organic fraction of municipal solid waste (OFMSW) in the overall kinetics of dry (30% total solids) thermophilic (55 Degree-Sign C) anaerobic digestion have been studied in a semi-continuous stirred tank reactor (SSTR). Two types of wastes were used: synthetic OFMSW (average particle size of 1 mm; 0.71 g Volatile Solids/g waste), and OFMSW coming from a composting full scale plant (average particle size of 30 mm; 0.16 g Volatile Solids/g waste). A modification of a widely-validated product-generation kinetic model has been proposed. Results obtained from the modified-model parameterization at steady-state (that include new kinetic parameters as K, Y{sub pMAX} and {theta}{sub MIN}) indicate that the features of the feedstock strongly influence the kinetics of the process. The overall specific growth rate of microorganisms ({mu}{sub max}) with synthetic OFMSW is 43% higher compared to OFMSW coming from a composting full scale plant: 0.238 d{sup -1} (K = 1.391 d{sup -1}; Y{sub pMAX} = 1.167 L CH{sub 4}/gDOC{sub c}; {theta}{sub MIN} = 7.924 days) vs. 0.135 d{sup -1} (K = 1.282 d{sup -1}; Y{sub pMAX} = 1.150 L CH{sub 4}/gDOC{sub c}; {theta}{sub MIN} = 9.997 days) respectively. Finally, it could be emphasized that the validation of proposed modified-model has been performed successfully by means of the simulation of non-steady state data for the different SRTs tested with each waste.

  5. Draft Genome Sequence of the Deinococcus-Thermus Bacterium Meiothermus ruber Strain A

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Thiel, Vera; Tomsho, Lynn P.; Burhans, Richard; Gay, Scott E.; Schuster, Stephan C.; Ward, David M.; Bryant, Donald A.

    2015-03-26

    The draft genome sequence of the Deinococcus-Thermus group bacterium Meiothermus ruber strain A, isolated from a cyanobacterial enrichment culture obtained from Octopus Spring (Yellowstone National Park, WY), comprises 2,968,099 bp in 170 contigs. It is predicted to contain 2,895 protein-coding genes, 44 tRNA-coding genes, and 2 rRNA operons.

  6. Functional and structural diversity in GH62 α-L-arabinofuranosidases from the thermophilic fungus Scytalidium thermophilum

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Kaur, Amrit Pal; Nocek, Boguslaw P.; Xu, Xiaohui; Lowden, Michael J.; Leyva, Juan Francisco; Stogios, Peter J.; Cui, Hong; Leo, Rosa Di; Powlowski, Justin; Tsang, Adrian; et al

    2015-05-01

    The genome of the thermophilic fungus Scytalidium thermophilum (strain CBS 625.91) harbours a wide range of genes involved in carbohydrate degradation, including three genes, abf62A, abf62B and abf62C, predicted to encode glycoside hydrolase family 62 (GH62) enzymes. Transcriptome analysis showed that only abf62A and abf62C are actively expressed during growth on diverse substrates including straws from barley, alfalfa, triticale and canola. The abf62A and abf62C genes were expressed in Escherichia coli and the resulting recombinant proteins were characterized. Calcium-free crystal structures of Abf62C in apo and xylotriose bound forms were determined to 1.23 and 1.48 Å resolution respectively. Site-directed mutagenesismore » confirmed Asp55, Asp171 and Glu230 as catalytic triad residues, and revealed the critical role of non-catalytic residues Asp194, Trp229 and Tyr338 in positioning the scissile α-L-arabinofuranoside bond at the catalytic site. Further, the +2R substrate-binding site residues Tyr168 and Asn339, as well as the +2NR residue Tyr226, are involved in accommodating long-chain xylan polymers. Overall, our structural and functional analysis highlights characteristic differences between Abf62A and Abf62C, which represent divergent subgroups in the GH62 family.« less

  7. A bacterium that can grow by using arsenic instead of phosphorus

    SciTech Connect (OSTI)

    Wolfe-Simon, F; Blum, J S; Kulp, T R; Gordon, G W; Hoeft, S E; Pett-Ridge, J; Stolz, J F; Webb, S M; Weber, P K; Davies, P W; Anbar, A D; Oremland, R S

    2010-11-01

    Life is mostly composed of the elements carbon, hydrogen, nitrogen, oxygen, sulfur and phosphorus. Although these six elements make up nucleic acids, proteins and lipids and thus the bulk of living matter, it is theoretically possible that some other elements in the periodic table could serve the same functions. Here we describe a bacterium, strain GFAJ-1 of the Halomonadaceae, isolated from Mono Lake, CA, which substitutes arsenic for phosphorus to sustain its growth. Our data show evidence for arsenate in macromolecules that normally contain phosphate, most notably nucleic acids and proteins. Exchange of one of the major bio-elements may have profound evolutionary and geochemical significance.

  8. Functional and structural diversity in GH62 ?-L-arabinofuranosidases from the thermophilic fungus Scytalidium thermophilum

    SciTech Connect (OSTI)

    Kaur, Amrit Pal; Nocek, Boguslaw P.; Xu, Xiaohui; Lowden, Michael J.; Leyva, Juan Francisco; Stogios, Peter J.; Cui, Hong; Leo, Rosa Di; Powlowski, Justin; Tsang, Adrian; Savchenko, Alexei

    2015-05-01

    The genome of the thermophilic fungus Scytalidium thermophilum (strain CBS 625.91) harbours a wide range of genes involved in carbohydrate degradation, including three genes, abf62A, abf62B and abf62C, predicted to encode glycoside hydrolase family 62 (GH62) enzymes. Transcriptome analysis showed that only abf62A and abf62C are actively expressed during growth on diverse substrates including straws from barley, alfalfa, triticale and canola. The abf62A and abf62C genes were expressed in Escherichia coli and the resulting recombinant proteins were characterized. Calcium-free crystal structures of Abf62C in apo and xylotriose bound forms were determined to 1.23 and 1.48 resolution respectively. Site-directed mutagenesis confirmed Asp55, Asp171 and Glu230 as catalytic triad residues, and revealed the critical role of non-catalytic residues Asp194, Trp229 and Tyr338 in positioning the scissile ?-L-arabinofuranoside bond at the catalytic site. Further, the +2R substrate-binding site residues Tyr168 and Asn339, as well as the +2NR residue Tyr226, are involved in accommodating long-chain xylan polymers. Overall, our structural and functional analysis highlights characteristic differences between Abf62A and Abf62C, which represent divergent subgroups in the GH62 family.

  9. Complete genome sequence of Anaeromyxobacter sp. Fw109-5, an anaerobic, metal-reducing bacterium isolated from a contaminated subsurface environment

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Hwang, C.; Copeland, A.; Lucas, S.; Lapidus, A.; Barry, K.; Glavina del Rio, T.; Dalin, E.; Tice, H.; Pitluck, S.; Sims, D.; et al

    2015-01-22

    We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacteriums genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation.

  10. Complete genome sequence of Anaeromyxobacter sp. Fw109-5, an Anaerobic, Metal-Reducing Bacterium Isolated from a Contaminated Subsurface Environment

    SciTech Connect (OSTI)

    Hwang, C.; Copeland, A.; Lucas, Susan; Lapidus, Alla; Barry, Kerrie W.; Glavina del Rio, T.; Dalin, Eileen; Tice, Hope; Pitluck, S.; Sims, David R.; Brettin, T.; Bruce, David; Detter, J. C.; Han, Cliff F.; Schmutz, Jeremy; Larimer, F.; Land, M.; Hauser, L.; Kyrpides, Nikos C.; Lykidis, Athanasios; Richardson, P. M.; Beliaev, Alex S.; Sanford, Robert A.; Loeffler, Frank E.; Fields, Matthew W.

    2015-01-22

    We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacteriums genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation.

  11. Near-complete genome sequence of the cellulolytic Bacterium Bacteroides (Pseudobacteroides) cellulosolvens ATCC 35603

    SciTech Connect (OSTI)

    Dassa, Bareket; Utturkar, Sagar M.; Hurt, Richard A.; Klingeman, Dawn Marie; Keller, Martin; Xu, Jian; Reddy, Harish Kumar; Borovok, Ilya; Grinberg, Inna Rozman; Lamed, Raphael; Zhivin, Olga; Bayer, Edward A.; Brown, Steven D.

    2015-09-24

    We report the single-contig genome sequence of the anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens. The bacterium produces a particularly elaborate cellulosome system, whereas the types of cohesin-dockerin interactions are opposite of other known cellulosome systems: cell-surface attachment is thus mediated via type-I interactions whereas enzymes are integrated via type-II interactions.

  12. Preliminary investigation of the effects of mineralogy and fluid composition on the growth of thermophilic bacteria in geothermal hot springs on the island of Vulcano, Italy

    SciTech Connect (OSTI)

    Amend, J.P.; Helgeson, H.C. . Dept. of Geology and Geophysics); Gurrieri, S.; Valenza, M. ); Clark, D.S. . Dept. of Chemical Engineering)

    1992-01-01

    Hydrothermal experiments were carried out recently on the island of Vulcano to investigate at in situ temperatures the relation of thermophilic bacterial growth to the mineralogy and fluid chemistry of geothermal hot springs. A preheated nutrient medium was inoculated with geothermal fluid and placed in the hydrothermal reactor, together with a sample of the mineralogic matrix through which the fluid flows. The results of the experiments are somewhat equivocal owing to (1) the inability to maintain the pH of the reactor fluid at the in situ pH (2.9 at 98 C), (2) apparent phase separation of what is probably a CO[sub 2]-rich gas leading to abnormally high pressures as the reactor temperature was increased in stages to 125 C, and (3) the fact that (unexpectedly) all of the bacteria were found to occur on the surfaces of mineral grains, which could not be sequentially collected in a representative manner with the apparatus at hand. Nevertheless, it appeared qualitatively that the population of bacteria increased during the experiment. Although this observation requires future confirmation and quantification with a more sophisticated reactor, the experimental results clearly indicate that conventional microbiological growth experiments using thermophilic bacteria that have been removed from their natural nutrient, in situ pH, and mineralogic environment may have little to do with the behavior of such bacteria in geothermal systems. Understanding this behavior requires integrated studies of the organobiogeochemistry of geothermal systems.

  13. Draft Genome Sequence for Microbacterium laevaniformans Strain OR221, a Bacterium Tolerant to Metals, Nitrate, and Low pH

    SciTech Connect (OSTI)

    Brown, Steven D; Palumbo, Anthony Vito; Panikov, Nikolai; Ariyawansa, Thilini; Klingeman, Dawn Marie; Johnson, Courtney M; Land, Miriam L; Utturkar, Sagar M; Epstein, Slava

    2012-01-01

    Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals such as uranium, nickel, cobalt, cadmium, as well as nitrate and low pH. We present its draft genome sequence.

  14. Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11

    SciTech Connect (OSTI)

    Yuki Kasai; Yumiko Kodama; Yoh Takahata; Toshihiro Hoaki; Kazuya Watanabe

    2007-09-15

    Azoarcus sp. strain DN11 is a denitrifying bacterium capable of benzene degradation under anaerobic conditions. The present study evaluated strain DN11 for its application to bioaugmentation of benzene-contaminated underground aquifers. Strain DN11 could grow on benzene, toluene, m-xylene, and benzoate as the sole carbon and energy sources under nitrate-reducing conditions, although o- and p-xylenes were transformed in the presence of toluene. Phenol was not utilized under anaerobic conditions. Kinetic analysis of anaerobic benzene degradation estimated its apparent affinity and inhibition constants to be 0.82 and 11 {mu}M, respectively. Benzene-contaminated groundwater taken from a former coal-distillation plant site in Aichi, Japan was anaerobically incubated in laboratory bottles and supplemented with either inorganic nutrients (nitrogen, phosphorus, and nitrate) alone, or the nutrients plus strain DN11, showing that benzene was significantly degraded only when DN11 was introduced. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments, and quantitative PCR revealed that DN11 decreased after benzene was degraded. Following the decrease in DN11 16S rRNA gene fragments corresponding to bacteria related to Owenweeksia hongkongensis and Pelotomaculum isophthalicum, appeared as strong bands, suggesting possible metabolic interactions in anaerobic benzene degradation. Results suggest that DN11 is potentially useful for degrading benzene that contaminates underground aquifers at relatively low concentrations. 50 refs., 6 figs., 1 tab.

  15. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    SciTech Connect (OSTI)

    Simpson, Philippa J.L.; Codd, Rachel; School of Medical Sciences and Bosch Institute, University of New South Wales, New South Wales 2006

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. Black-Right-Pointing-Pointer Protein homology model of NapA from S. gelidimarina and mesophilic homologue. Black-Right-Pointing-Pointer Six amino acid residues identified as lead candidates governing NapA cold adaptation. Black-Right-Pointing-Pointer Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap{sub Sgel}) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap{sub Sput}) was examined at varied temperature. Irreversible deactivation of Nap{sub Sgel} and Nap{sub Sput} occurred at 54.5 and 65 Degree-Sign C, respectively. When Nap{sub Sgel} was preincubated at 21-70 Degree-Sign C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 Degree-Sign C, which suggested that Nap{sub Sgel} was poised for optimal catalysis at modest temperatures and, unlike Nap{sub Sput}, did not benefit from thermally-induced refolding. At 20 Degree-Sign C, Nap{sub Sgel} reduced selenate at 16% of the rate of nitrate reduction. Nap{sub Sput} did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap{sub Sgel} that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap{sub Sgel} cold-adapted phenotype. Protein homology modeling of Nap{sub Sgel} using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo-MGD cofactor. Two mesophilic {r_reversible} psychrophilic substitutions (Asn {r_reversible} His, Val {r_reversible} Trp) occurred in a region close to the surface of the NapA substrate funnel resulting in potential interdomain {pi}-{pi} and/or cation-{pi} interactions. Three mesophilic {r_reversible} psychrophilic substitutions occurred within 4.5 A of the Mo-MGD cofactor (Phe {r_reversible} Met, Ala {r_reversible} Ser, Ser {r_reversible} Thr) resulting in local regions that varied in hydrophobicity and hydrogen bonding networks. These results contribute to the understanding of thermal protein adaptation in a redox-active mononuclear molybdenum enzyme and have implications in optimizing the design of low-temperature environmental biosensors.

  16. Restriction/modification polypeptides, polynucleotides, and methods

    DOE Patents [OSTI]

    Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

    2015-02-24

    The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

  17. Genome sequence of the thermophilic fresh-water bacterium Spirochaeta caldaria type strain (H1T), reclassification of Spirochaeta caldaria, Spirochaeta stenostrepta, and Spirochaeta zuelzerae in the genus Treponema as Treponema caldaria comb. nov., Treponema stenostrepta comb. nov., and Treponema zuelzerae comb. nov., and emendation of the genus Tr

    SciTech Connect (OSTI)

    Abt, Birte; Goker, Markus; Scheuner, Carmen; Han, Cliff; Lu, Megan; Misra, Monica; Lapidus, Alla L.; Nolan, Matt; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxanne; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Pagani, Ioanna; Ivanova, N; Mavromatis, K; Mikhailova, Natalia; Huntemann, Marcel; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Rohde, Manfred; Spring, Stefan; Gronow, Sabine; Detter, J. Chris; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Woyke, Tanja; Klenk, Hans-Peter

    2013-01-01

    Spirochaeta caldaria Pohlschroeder et al. 1995 is an obligately anaerobic, spiral-shaped bac- terium that is motile via periplasmic flagella. The type strain, H1T, was isolated in 1990 from cyanobacterial mat samples collected at a freshwater hot spring in Oregon, USA, and is of in- terest because it enhances the degradation of cellulose when grown in co-culture with Clos- tridium thermocellum. Here we provide a taxonomic re-evaluation for S. caldaria based on phylogenetic analyses of 16S rRNA sequences and whole genomes, and propose the reclassi- fication of S. caldaria and two other Spirochaeta species as members of the emended genus Treponema. Whereas genera such as Borrelia and Sphaerochaeta possess well-distinguished genomic features related to their divergent lifestyles, the physiological and functional ge- nomic characteristics of Spirochaeta and Treponema appear to be intermixed and are of little taxonomic value. The 3,239,340 bp long genome of strain H1T with its 2,869 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  18. Kallotenue papyrolyticum gen. nov., sp. nov., a cellulolytic and filamentous thermophile that represents a novel lineage (Kallotenuales ord. nov., Kallotenuaceae fam. nov.) within the class Chloroflexia

    SciTech Connect (OSTI)

    Cole, Jesse; Gieler, Brandon; Heisler, Devon; Palisoc, Maryknoll; Williams, Amanda; Dohnalkova, Alice; Ming, Hong; Yu, Tian T.; Dodsworth, Jeremy A.; Li, Wen J.; Hedlund, Brian P.

    2013-08-15

    Several closely-related, thermophilic, and cellulolytic bacterial strains, designated JKG1T, JKG2, JKG3, JKG4, and JKG5, were isolated from a cellulolytic enrichment (corn stover) incubated in the water column of Great Boiling Spring, NV. Strain JKG1T had cells of a diameter of 0.7 - 0.9 ?m and length of ~2.0 ?m that formed non-branched multicellular filaments reaching >300 ?m. Spores were not formed and dense liquid cultures were red. The temperature range for growth was 45-65 C, with an optimum of 55 C. The pH range for growth was 5.6-9.0, with an optimum of 7.5. JKG1T grew as an aerobic heterotroph, utilizing glucose, sucrose, xylose, arabinose, cellobiose, carboxymethylcellulose, filter paper, microcrystalline cellulose, xylan, starch, casamino acids, tryptone, peptone, yeast extract, acetate, citrate, lactate, pyruvate, and glycerol as sole carbon sources, and was not observed to photosynthesize. The cells stained Gram-negative. Phylogenetic analysis using 16S rRNA gene sequences placed the new isolates in the class Chloroflexia, but distant from other cultivated members, with the highest sequence identity of 82.5% to Roseiflexus castenholzii. The major quinone was menaquinone-9; no ubiquinones were detected. The major cellular fatty acids (>5%) were C18:0, anteiso-C17:0, iso-C18:0, and iso-C17:0. C16:0, iso-C16:0, and C17:0. The peptidoglycan amino acids were alanine, ornithine, glutamic acid, serine, and asparagine. Whole-cell sugars included mannose, rhamnose, glucose, galactose, ribose, arabinose, and xylose. Morphological, phylogenetic, and chemotaxonomic results suggest that JKG1T is representative of a new lineage within the class Chloroflexia, which we propose to designate Kallotenue papyrolyticum gen. nov., sp. nov., Kallotenuaceae fam. nov., Kallotenuales ord. nov.

  19. Complete genome sequence of Thioalkalivibrio paradoxus type strain ARh 1T, an obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacterium isolated from a Kenyan soda lake

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Berben, Tom; Sorokin, Dimitry Y.; Ivanova, Natalia; Pati, Amrita; Kyrpides, Nikos; Goodwin, Lynne A.; Woyke, Tanja; Muyzer, Gerard

    2015-11-19

    Thioalkalivibrio paradoxus strain ARh 1T is a chemolithoautotrophic, non-motile, Gram-negative bacterium belonging to the Gammaproteobacteria that was isolated from samples of haloalkaline soda lakes. It derives energy from the oxidation of reduced sulfur compounds and is notable for its ability to grow on thiocyanate as its sole source of electrons, sulfur and nitrogen. The full genome consists of 3,756,729 bp and comprises 3,500 protein-coding and 57 RNA-coding genes. Moreover, this organism was sequenced as part of the community science program at the DOE Joint Genome Institute.

  20. Crystallization and preliminary X-ray analysis of a class II release factor RF3 from a sulfate-reducing bacterium

    SciTech Connect (OSTI)

    Kihira, Kiyohito; Numata, Shuko; Kitamura, Masaya; Kondo, Jun; Terawaki, Shinichi; Shomura, Yasuhito; Komori, Hirofumi; Shibata, Naoki; Higuchi, Yoshiki

    2008-07-01

    Class II release factor 3 (RF3) from the sulfate-reducing bacterium D. vulgaris Miyazaki F has been overexpressed, purified and crystallized in complex with GDP. Class II release factor 3 (RF3) from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F, which promotes rapid dissociation of a class I release factor, has been overexpressed, purified and crystallized in complex with GDP at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to 1.8 resolution from a single crystal at 100 K using synchrotron radiation. The crystal belongs to space group P1, with unit-cell parameters a = 47.39, b = 82.80, c = 148.29 , ? = 104.21, ? = 89.78, ? = 89.63. The asymmetric unit contains four molecules of the RF3GDP complex. The Matthews coefficient was calculated to be 2.3 {sup 3} Da{sup ?1} and the solvent content was estimated to be 46.6%.

  1. evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation ans a consolidated bioprocessing approach

    SciTech Connect (OSTI)

    Yee, Kelsey L; Rodriguez, Jr., Miguel; Tschaplinski, Timothy J; Engle, Nancy L; Martin, Madhavi Z; Fu, Chunxiang; Wang, Zeng-Yu; Hamilton-Brehm, Scott; Mielenz, Jonathan R

    2012-01-01

    Abstract Background: The inherent recalcitrance of lignocellulosic biomass is one of the major economic hurdles for the production of fuels and chemicals from biomass. Additionally, lignin is recognized as having a negative impact on enzymatic hydrolysis of biomass, and as a result much interest has been placed on modifying the lignin pathway to improve bioconversion of lignocellulosic feedstocks. Results: Previous results showed down-regulation of the caffeic acid 3-O-methyl transferase (COMT) gene in the lignin pathway yielded switchgrass (Panicum virgatum) that was more susceptible to bioconversion after dilute acid pretreatment. Here we examined the response of these plant lines to milder pretreatment conditions with yeast-based SSF, CBP with Clostridium thermocellum, and fermentations with the cellulolytic extreme thermophiles, Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis. Unlike the S. cerevisiae SSF conversions, fermentations of pretreated down-regulated COMT transgenic switchgrass with C. thermocellum showed an apparent inhibition of fermentation not observed in the wild-type switchgrass. This inhibition can be eliminated by hot water extraction of the pretreated biomass which resulted in superior conversion yield with transgenic versus wild-type switchgrass for C. thermocellum, also exceeding the yeast-based SSF yield. Further fermentation evaluation of the transgenic switchgrass indicated differential inhibition for the Caldicellulosiruptor strains, which could not be rectified by additional processing conditions. Gas chromatography-mass spectrometry metabolite profiling was used to examine the fermentation broth to elucidate the relative abundance of lignin derived aromatic compounds. The types and abundance of fermentation-derived lignin constituents varied between C. thermocellum and each of the Caldicellulosiruptor strains. Conclusions: The down-regulation of the COMT gene improves the bioconversion of switchgrass relative to the wild-type regardless of the pretreatment condition or fermentation microorganism. However, bacterial fermentations demonstrated strain-dependent sensitivity to the COMT transgenic biomass, likely due to additional soluble lignin pathway-derived constituents resulting from the COMT gene disruption. Removal of these inhibitory constituents permitted completion of fermentation by C. thermocellum, but not by the Caldicellulosiruptor strains. The reason for this difference needs to be explored further.

  2. Genome Sequence of the Mesophilic Thermotogales Bacterium Mesotoga prima MesG1.Ag.4.2 Reveals the Largest Thermotogales Genome To Date

    SciTech Connect (OSTI)

    Zhaxybayeva, Olga; Swithers, Kristen S; Foght, Julia; Green, Anna G.; Bruce, David; Detter, J. Chris; Han, Cliff; Teshima, Hazuki; Han, James; Woyke, Tanja; Pitluck, Sam; Nolan, Matt; Ivanova, N; Pati, Amrita; Land, Miriam L; Dlutek, Marlena; Doolittle, W. Ford; Noll, Kenneth M; Nesbo, Camilla

    2012-01-01

    Here we describe the genome of Mesotoga prima MesG1.Ag4.2, the first genome of a mesophilic Thermotogales bacterium. Mesotoga prima was isolated from a polychlorinated biphenyl (PCB)-dechlorinating enrichment culture from Baltimore Harbor sediments. Its 2.97 Mb genome is considerably larger than any previously sequenced Thermotogales genomes, which range between 1.86 and 2.30 Mb. This larger size is due to both higher numbers of protein-coding genes and larger intergenic regions. In particular, the M. prima genome contains more genes for proteins involved in regulatory functions, for instance those involved in regulation of transcription. Together with its closest relative, Kosmotoga olearia, it also encodes different types of proteins involved in environmental and cell-cell interactions as compared with other Thermotogales bacteria. Amino acid composition analysis of M. prima proteins implies that this lineage has inhabited low-temperature environments for a long time. A large fraction of the M. prima genome has been acquired by lateral gene transfer (LGT): a DarkHorse analysis suggests that 766 (32%) of predicted protein-coding genes have been involved in LGT after Mesotoga diverged from the other Thermotogales lineages. A notable example of a lineage-specific LGT event is a reductive dehalogenase gene - a key enzyme in dehalorespiration, indicating M. prima may have a more active role in PCB dechlorination than was previously assumed.

  3. Ultra-broadband 2D electronic spectroscopy of carotenoid-bacteriochlorophyll interactions in the LH1 complex of a purple bacterium

    SciTech Connect (OSTI)

    Maiuri, Margherita; Rhault, Julien; Polli, Dario; Cerullo, Giulio; Carey, Anne-Marie; Hacking, Kirsty; Cogdell, Richard J.; Garavelli, Marco; Ler, Larry

    2015-06-07

    We investigate the excitation energy transfer (EET) pathways in the photosynthetic light harvesting 1 (LH1) complex of purple bacterium Rhodospirillum rubrum with ultra-broadband two-dimensional electronic spectroscopy (2DES). We employ a 2DES apparatus in the partially collinear geometry, using a passive birefringent interferometer to generate the phase-locked pump pulse pair. This scheme easily lends itself to two-color operation, by coupling a sub-10 fs visible pulse with a sub-15-fs near-infrared pulse. This unique pulse combination allows us to simultaneously track with extremely high temporal resolution both the dynamics of the photoexcited carotenoid spirilloxanthin (Spx) in the visible range and the EET between the Spx and the B890 bacterio-chlorophyll (BChl), whose Q{sub x} and Q{sub y} transitions peak at 585 and 881 nm, respectively, in the near-infrared. Global analysis of the one-color and two-color 2DES maps unravels different relaxation mechanisms in the LH1 complex: (i) the initial events of the internal conversion process within the Spx, (ii) the parallel EET from the first bright state S{sub 2} of the Spx towards the Q{sub x} state of the B890, and (iii) the internal conversion from Q{sub x} to Q{sub y} within the B890.

  4. Comparative proteomic analysis of Desulfotomaculum reducens MI-1: Insights into the metabolic versatility of a gram-positive sulfate- and metal-reducing bacterium

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Otwell, Anne E.; Callister, Stephen J.; Zink, Erika M.; Smith, Richard D.; Richardson, Ruth E.

    2016-02-19

    In this study, the proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism cultivated either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfatemore » (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals.« less

  5. Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

    SciTech Connect (OSTI)

    Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng; Nelson, Ornella D.; Li, Zhi; Lin, Hening; Callister, Stephen J.; Richardson, Ruth E.

    2015-01-01

    Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor, we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.

  6. The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: A blueprint for thriving in and out of symbiosis

    SciTech Connect (OSTI)

    Dmytrenko, Oleg; Russell, Shelbi L.; Loo, Wesley T.; Fontanez, Kristina M.; Liao, Li; Roeselers, Guus; Sharma, Raghav; Stewart, Frank J.; Newton, Irene L. G.; Woyke, Tanja; Wu, Dongying; Lang, Jenna Morgan; Eisen, Jonathan A.; Cavanaugh, Colleen M.

    2014-09-25

    Background: Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum, and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions. To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced. Results: Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.86 Mb), GC-rich (50.4percent), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host. Conclusions: The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle

  7. Complete genome sequence of Thioalkalivibrio paradoxus type strain ARh 1T, an obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacterium isolated from a Kenyan soda lake

    SciTech Connect (OSTI)

    Berben, Tom; Sorokin, Dimitry Y.; Ivanova, Natalia; Pati, Amrita; Kyrpides, Nikos; Goodwin, Lynne A.; Woyke, Tanja; Muyzer, Gerard

    2015-11-19

    Thioalkalivibrio paradoxus strain ARh 1T is a chemolithoautotrophic, non-motile, Gram-negative bacterium belonging to the Gammaproteobacteria that was isolated from samples of haloalkaline soda lakes. It derives energy from the oxidation of reduced sulfur compounds and is notable for its ability to grow on thiocyanate as its sole source of electrons, sulfur and nitrogen. The full genome consists of 3,756,729 bp and comprises 3,500 protein-coding and 57 RNA-coding genes. Moreover, this organism was sequenced as part of the community science program at the DOE Joint Genome Institute.

  8. Thermophilic Endoglucanase Enzymes Engineered for Increased Activity...

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Contact LBL About This Technology Technology Marketing SummaryResearchers at the Joint BioEnergy Institute (JBEI) have generated and identified new enzyme variants of cellulase ...

  9. Thermophilic Cellulases Compatible with Ionic Liquid Pretreatment...

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Ionic liquids dissolve cellulose, which can then be separated out in an additional process. However, significant decreases in the available commercial fungal cellulase activity in ...

  10. Novel Thermophilic Cellobiohydrolase - Energy Innovation Portal

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Higher activity, longer duration and greater tolerance to ionic liquids compared to commercially available cellulase cocktails Facilitates one pot pretreatmentsaccharification of ...

  11. Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1 T ), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaeta

    SciTech Connect (OSTI)

    Abt, Birte; Han, Cliff; Scheuner, Carmen; Lu, Megan; Lapidus, Alla L.; Nolan, Matt; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxanne; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Pagani, Ioanna; Ivanova, N; Mavromatis, K; Mikhailova, Natalia; Huntemann, Marcel; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Hauser, Loren John; Brambilla, Evelyne-Marie; Rohde, Manfred; Spring, Stefan; Gronow, Sabine; Goker, Markus; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Detter, J. Chris

    2012-01-01

    Spirochaeta coccoides Droege et al. 2006 is a member of the genus Spirochaeta Ehrenberg 1835, one of the oldest named genera within the Bacteria. S. coccoides is an obligately anaerobic, Gram-negative, non-motile, spherical bacterium that was isolated from the hindgut contents of the termite Neotermes castaneus. The species is of interest because it may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut. Here we provide a taxonomic re-evaluation for strain SPN1{sup T}, and based on physiological and genomic characteristics, we propose its reclassification as a novel species in the genus Sphaerochaeta, a recently published sister group of the Spirochaeta. The 2,227,296 bp long genome of strain SPN1{sup T} with its 1,866 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  12. Draft Genome Sequences for Clostridium thermocellum Wild-Type Strain YS and Derived Cellulose Adhesion-Defective Mutant Strain AD2

    SciTech Connect (OSTI)

    Brown, Steven D; Lamed, Raphael; Morag, Ely; Borovok, Ilya; Shoham, Yuval; Klingeman, Dawn Marie; Johnson, Courtney M; Yang, Zamin; Land, Miriam L; Utturkar, Sagar M; Keller, Martin; Bayer, Edward A

    2012-01-01

    Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain AD2 played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

  13. Promiscuous Plasmid Replication in Thermophiles: Use of a Novel...

    Office of Scientific and Technical Information (OSTI)

    Additional Journal Information: Journal Volume: 3; Related Information: Metabolic Engineering Communications; Journal ID: ISSN 2214-0301 Publisher: Elsevier Research Org: NREL ...

  14. Microbial Ecology of Thermophilic Anaerobic Digestion. Final Report

    DOE R&D Accomplishments [OSTI]

    Zinder, Stephen H.

    2000-04-15

    This grant supported research on methanogenic archaea. The two major areas that were supported were conversion of acetic acid to methane and nitrogen fixation by Methanosarcina. Among the achievements of this research were the isolation of novel methanogenic cultures, elucidation of the pathways from acetate to methane, description of a specific DNA-binding complex in nitrogen fixing methanogens, and demonstration of an alternative nitrogenase in Methanosarcina.

  15. Development of a thermophilic SSF system for butanol production...

    Office of Energy Efficiency and Renewable Energy (EERE) Indexed Site

    chemical intermediate from all sugars present - Integration and Intensification - ... conversion process 3 | Bioenergy ... focus on genetic engineering of fermentation ...

  16. Microbial ecology of thermophilic anaerobic digestion. Final report

    SciTech Connect (OSTI)

    Stephen H. Zinder

    2000-04-15

    This grant supported research on methanogenic archaea. The two major areas that were supported were conversion of acetic acid to methane and nitrogen fixation by Methanosarcina. Among the achievements of this research were the isolation of novel methanogenic cultures, elucidation of the pathways from acetate to methane, description of a specific DNA-binding complex in nitrogen fixing methanogens, and demonstration of an alternative nitrogenase in Methanosarcina.

  17. Fermentation method producing ethanol

    DOE Patents [OSTI]

    Wang, Daniel I. C.; Dalal, Rajen

    1986-01-01

    Ethanol is the major end product of an anaerobic, thermophilic fermentation process using a mutant strain of bacterium Clostridium thermosaccharolyticum. This organism is capable of converting hexose and pentose carbohydrates to ethanol, acetic and lactic acids. Mutants of Clostridium thermosaccharolyticum are capable of converting these substrates to ethanol in exceptionally high yield and with increased productivity. Both the mutant organism and the technique for its isolation are provided.

  18. Composition of the cellulase complex of clostridium thermocellum

    SciTech Connect (OSTI)

    Golovchenko, N.P.; Akimenko, V.K.; Chuvil'skaya, N.A.

    1985-07-20

    The anaerobic thermophilic cellulolytic bacterium C. thermocellum is considered as a potential organism for the industrial direct bioconversion of cellulose to ethanol. The activities and localization of various cellulolytic enzymes in C. thermocellum are determined here. Preparative methods and determination of enzymes are discussed. Analytical methods are examined. The authors determine that C. thermocellum has all six known cellulolytic enzymes, and that in certain cases up to 50% of the intracellular activity of the indicated enzymes is localized outside the cells.

  19. Genetic manipulation of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans

    SciTech Connect (OSTI)

    Beller, H.R.; Legler, T.C.; Kane, S.R.

    2011-07-15

    Chemolithoautotrophic bacteria can be of industrial and environmental importance, but they present a challenge for systems biology studies, as their central metabolism deviates from that of model organisms and there is a much less extensive experimental basis for their gene annotation than for typical organoheterotrophs. For microbes with sequenced genomes but unconventional metabolism, the ability to create knockout mutations can be a powerful tool for functional genomics and thereby render an organism more amenable to systems biology approaches. In this chapter, we describe a genetic system for Thiobacillus denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. Insertion mutations are generated by in vitro transposition, the mutated genes are amplified by the PCR, and the amplicons are introduced into T. denitrificans by electroporation. Use of a complementation vector, pTL2, based on the IncP plasmid pRR10 is also addressed.

  20. Process for generation of hydrogen gas from various feedstocks using thermophilic bacteria

    DOE Patents [OSTI]

    Ooteghem, Suellen Van

    2005-09-13

    A method for producing hydrogen gas is provided comprising selecting a bacteria from the Order Thermotogales, subjecting the bacteria to a feedstock and to a suitable growth environment having an oxygen concentration below the oxygen concentration of water in equilibrium with air; and maintaining the environment at a predetermined pH and at a temperature of at least approximately 45.degree. C. for a time sufficient to allow the bacteria to metabolize the feedstock.

  1. Thermophilic and thermoacidophilic sugar transporter genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2011-06-14

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  2. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-07-30

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  3. Thermophilic and thermoacidophilic metabolism genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, Vicki S.; Apel, William A.; Reed, David William; Lee, Brady D.; Thompson, David N.; Roberto, Francisco F.; Lacey, Jeffrey A.

    2015-12-29

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  4. Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A

    2013-11-05

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  5. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2012-06-19

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  6. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2015-06-02

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  7. Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2011-12-06

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  8. Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2016-01-12

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for glycosylating and/or post-translationally modifying proteins using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  9. Thermophilic and thermoacidophilic metabolism genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, Vicki S; Apel, William A; Reed, David W; Lee, Brady D; Thompson, David N; Roberto, Francisco F; Lacey, Jeffrey A

    2014-05-20

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  10. Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2013-01-29

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  11. Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2013-01-15

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  12. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-04-23

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  13. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2013-10-15

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  14. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2010-12-28

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  15. Scalable economic extracellular synthesis of CdS nanostructured particles by a non-pathogenic thermophile

    SciTech Connect (OSTI)

    Moon, Ji Won; Ivanov, Ilia N; Duty, Chad E; Love, Lonnie J; Rondinone, Adam Justin; Wang, Wei; Li, Dr. Yi-Liang; Madden, Andrew; Mosher, Jennifer J; Hu, Michael Z.; Suresh, Anil K; Rawn, Claudia J; Jung, Hyunsung; Lauf, Robert J; Phelps, Tommy Joe

    2013-01-01

    We report microbially facilitated synthesis of cadmium sulfide (CdS) nanostructured particles (NP) using anaerobic, metal-reducing Thermoanaerobacter sp. The extracellular CdS crystallites were <10 nm in size with yields of ~3 g/L of growth medium/month with demonstrated reproducibility and scalability up to 24 L. During synthesis, Thermoanaerobacter cultures reduced thiosulfate and sulfite salts to H2S, which reacted with Cd2+ cations to produce thermodynamically favored NP in a single step at 65oC with catalytic nucleation on the cell surfaces. Photoluminescence (PL) analysis of dry CdS NP revealed an exciton-dominated PL peak at 440 nm, having a narrow full width at half maximum of 10 nm. A PL spectrum of CdS NP produced by dissimilatory sulfur reducing bacteria was dominated by features associated with radiative exciton relaxation at the surface. High reproducibility of CdS NP PL features important for scale-up conditions was confirmed from test tubes to 24L batches at a small fraction of the manufacturing cost associated with conventional inorganic NP production processes.

  16. Bioenergetic studies of coal sulfur oxidation by extremely thermophilic bacteria. Final report, September 15, 1992--August 31, 1997

    SciTech Connect (OSTI)

    Kelly, R.M.; Han, C.J.

    1997-12-31

    Thermoacidophilic microorganisms have been considered for inorganic sulfur removal from coal because of expected improvements in rates of both biotic and abiotic sulfur oxidation reactions with increasing temperature. In this study, the bioenergetic response of the extremely thermoacidophilic archaeon, Metallosphaera sedula, to environmental changes have been examined in relation to its capacity to catalyze pyrite oxidation in coal. Given an appropriate bioenergetic challenge, the metabolic response was to utilize additional amounts of energy sources (i.e., pyrite) to survive. Of particular interest were the consequences of exposing the organism to various forms of stress (chemical, nutritional, thermal, pH) in the presence of coal pyrite. Several approaches to take advantage of stress response to accelerate pyrite oxidation by this organism were examined, including attempts to promote acquired thermal tolerance to extend its functional range, exposure to chemical uncouplers and decouplers, and manipulation of heterotrophic and chemolithotrophic tendencies to optimize biomass concentration and biocatalytic activity. Promising strategies were investigated in a continuous culture system. This study identified environmental conditions that promote better coupling of biotic and abiotic oxidation reactions to improve biosulfurization rates of thermoacidophilic microorganisms.

  17. Long-term thermophilic mono-digestion of rendering wastes and co-digestion with potato pulp

    SciTech Connect (OSTI)

    Bayr, S. Ojanper, M.; Kaparaju, P.; Rintala, J.

    2014-10-15

    Highlights: Rendering wastes mono-digestion and co-digestion with potato pulp were studied. CSTR process with OLR of 1.5 kg VS/m{sup 3} d, HRT of 50 d was unstable in mono-digestion. Free NH{sub 3} inhibited mono-digestion of rendering wastes. CSTR process with OLR of 1.5 kg VS/m{sup 3} d, HRT of 50 d was stable in co-digestion. Co-digestion increased methane yield somewhat compared to mono-digestion. - Abstract: In this study, mono-digestion of rendering wastes and co-digestion of rendering wastes with potato pulp were studied for the first time in continuous stirred tank reactor (CSTR) experiments at 55 C. Rendering wastes have high protein and lipid contents and are considered good substrates for methane production. However, accumulation of digestion intermediate products viz., volatile fatty acids (VFAs), long chain fatty acids (LCFAs) and ammonia nitrogen (NH{sub 4}-N and/or free NH{sub 3}) can cause process imbalance during the digestion. Mono-digestion of rendering wastes at an organic loading rate (OLR) of 1.5 kg volatile solids (VS)/m{sup 3} d and hydraulic retention time (HRT) of 50 d was unstable and resulted in methane yields of 450 dm{sup 3}/kg VS{sub fed}. On the other hand, co-digestion of rendering wastes with potato pulp (60% wet weight, WW) at the same OLR and HRT improved the process stability and increased methane yields (500680 dm{sup 3}/kg VS{sub fed}). Thus, it can be concluded that co-digestion of rendering wastes with potato pulp could improve the process stability and methane yields from these difficult to treat industrial waste materials.

  18. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOE Patents [OSTI]

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  19. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOE Patents [OSTI]

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  20. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOE Patents [OSTI]

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  1. Ethanol production by thermophilic bacteria: fermentation of cellulosic substrates by cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

    SciTech Connect (OSTI)

    Ng, T.K.; Ben-Bassat, A.; Zeikus, J.G.

    1981-06-01

    The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0 C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of approx. 1.0. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze ..cap alpha..-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate.

  2. Tropical Soil Bacterium Frees Plant Sugars for Biofuels | U.S...

    Office of Science (SC) Website

    As part of research to improve biofuel production processes, ... abundant, and nonfood energy source that could be used to make sustainable and economically feasible biofuels. ...

  3. Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

    SciTech Connect (OSTI)

    Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.; Blow, Matthew; Korlach, Jonas; Deutschbauer, Adam; Malmstrom, Rex

    2013-08-30

    We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns. However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.

  4. Evidence for Multiple Modes of Uranium Immobilization by an Anaerobic Bacterium

    SciTech Connect (OSTI)

    Allison E. Ray; John R. Bargar; Alice C. Dohnalkova; Vaidee Sivaswamy; Yoshiko Fujita; Timothy S. Magnuson

    2011-05-01

    ABSTRACT Microbial reduction of hexavalent uranium has been studied widely for its potential role in bioremediation and removal of soluble U(VI) from contaminated groundwater. More recently, some microorganisms have been examined for their role in immobilization of U(VI) via precipitation of uranyl phosphate minerals mediated by microbial phosphate release, alleviating the requirement for long-term redox control. Here, we investigated the mechanism of U(VI) removal mediated by an environmental isolate, strain UFO1, that is indigenous to the Field Research Center (FRC) in Oak Ridge, TN and has been detected in U(VI)-contaminated sediments. U(VI) removal was examined in the presence and absence of the electron-shuttling moiety, anthraquinone-2,6-disulfonate (AQDS). Cell suspensions were capable of the near complete removal of 100 uM U(VI) from solution within 48 hours; U(VI) removal was not dependent on the presence of an exogenous electron donor or AQDS, although AQDS increased the rate of U(VI) removal. Profiles of ortho-phosphate concentration over time suggested phosphate liberation from cells. However, X-ray Absorption Near Edge Structure (XANES) spectroscopic measurements indicated that U(IV) was the predominant oxidation state of uranium in cell suspensions in both the absence and presence of 100 uM AQDS. Extended X-ray Absorption Fine Structure spectroscopy (EXAFS) measurements indicated that 20% of the cell-associated precipitates in a U(VI)-treated suspension that lacked AQDS had spectral characteristics consistent with a uranyl phosphate solid phase. EXAFS fits further show that that U(IV) is present dominantly as a monomeric sorbed complex. TEM-EDS confirmed the presence of uranyl phosphate with a U:P ratio consistent with autunite (1:1). These results suggest that strain UFO1 has the ability to mediate U(VI) removal from solution via both reductive and phosphate precipitation mechanisms, and may potentially be useful for the remediation of U-contaminated sediments at the FRC.

  5. Evidence for Multiple Modes of Uranium Immobilization by an Anaerobic Bacterium

    SciTech Connect (OSTI)

    Ray, Allison; Bargar, John R.; Sivaswamy, Vaideeswaran; Dohnalkova, Alice; Fujita, Yoshiko; Peyton, Brent M.; Magnuson, Timothy S.

    2011-05-15

    Microbial reduction of hexavalent uranium has been studied widely for its potential role in bioremediation and immobilization of soluble U(VI) in contaminated groundwater. More recently, some microorganisms have been examined for their role in immobilization of U(VI) via precipitation of uranyl phosphate minerals mediated by microbial phosphate release, alleviating the requirement for long-term redox control. Here, we investigated the mechanism of U(VI) removal mediated by an environmental isolate, strain UFO1, that is indigenous to the Field Research Center (FRC) in Oak Ridge, TN and has been detected in U(VI)-contaminated sediments. Changes in U(VI) speciation were examined in the presence and absence of the electron-shuttling moiety, anthraquinone-2,6-disulfonate (AQDS). Cell suspensions were capable of nearly complete removal of 100 M U(VI) from solution within 48 hours; U(VI) removal was not dependent on the presence of an exogenous electron donor or AQDS, although AQDS increased the rate of U(VI) removal. X-ray Absorption Near Edge Structure (XANES) spectroscopic measurements indicated that U(IV) was the predominant oxidation state of uranium in cell suspensions in both the absence and presence of 100 M AQDS. However, extended X-ray Absorption Fine Structure spectroscopy (EXAFS) measurements indicated that 17% of the cell-associated precipitates in a U(VI)-treated suspension that lacked AQDS had spectral characteristics consistent with a uranyl phosphate solid phase. The potential involvement of phosphate was consistent with observed increases in soluble phosphate concentrations over time in UFO1 cell suspensions, which suggested phosphate liberation from the cells. TEM-EDS confirmed the presence of uranyl phosphate with a U:P ratio consistent with autunite (1:1). EXAFS analyses further showed that U(IV) was present predominantly as a monomeric complex sorbed to carboxylate functional groups on biomass and also suggested that a fraction of the U(IV) was coordinated to phosphoryl ligands. These results suggest that strain UFO1 has the ability to facilitate U(VI) removal from solution via both reductive and phosphate precipitation mechanisms, and may potentially be useful for the remediation of U-contaminated sediments at the FRC or elsewhere.

  6. Made Famous By Erin Brockovich: A Potent Pollutant and its Nemesis Bacterium

    Broader source: Energy.gov [DOE]

    This element, made famous in the settlement case won by Erin Brockovich, can now be tamed with a process created by the Brookhaven Lab.

  7. Proteolysis in hyperthermophilic microorganisms

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; Levy, Ryan D.; Michel, Joshua K.; Conners, Shannon B.; Kelly, Robert M.

    2002-01-01

    Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus , the crenarchaeote Sulfolobus solfataricus , and the bacterium Thermotoga maritima . An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putativemore » proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.« less

  8. Environmental genomics reveals a single species ecosystem deep within the Earth

    SciTech Connect (OSTI)

    Chivian, Dylan; Brodie, Eoin L.; Alm, Eric J.; Culley, David E.; Dehal, Paramvir S.; DeSantis, Todd Z.; Gihring, Thomas M.; Lapidus, Alla; Lin, Li-Hung; Lowry, Stephen R.; Moser, Duane P.; Richardson, Paul; Southam, Gordon; Wanger, Greg; Pratt, Lisa M.; Andersen, Gary L.; Hazen, Terry C.; Brockman, Fred J.; Arkin, Adam P.; Onstott, Tullis C.

    2008-09-17

    DNA from low biodiversity fracture water collected at 2.8 km depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, comprises>99.9percent of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent lifestyle well suited to long-term isolation from the photosphere deep within Earth?s crust, and offers the first example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

  9. Exchange of Type II Dockerin-Containing Subunits of the C. thermocellum Cellulosome as Revealed by SNAP-tags

    SciTech Connect (OSTI)

    Waller, Benjamin; Olson, Daniel G.; Currie, Devin; Guss, Adam M; Lynd, Lee R

    2013-01-01

    Clostridium thermocellum is a thermophilic anaerobic bacterium which efficiently hydrolyzes and metabolizes cellulose to ethanol through the action of its cellulosome, a multiprotein enzymatic complex. A fluorescent protein probe, consisting of a type II dockerin module fused to a SNAP-tag, was developed in order to gain insight into the quaternary configuration of the cellulosome and to investigate the effect of deleting cipA, the protein scaffold on which the cellulosome is built. Fluorescence microscopy suggested that the probe had localized to polycellulosomal protuberances on the cell surface. Surprisingly, fluorescence intensity did not substantially change in the cipA deletion mutants. Sequential labeling experiments suggested that this was a result of bound type II dockerins from CipA being replaced by unbound type II dockerins from the fluorophore-SNAP-XDocII probe. This mechanism of dockerin exchange could represent an efficient means for modifying cellulosome composition.

  10. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    SciTech Connect (OSTI)

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Bhandiwad, Ashwini; Rodriguez, Jr., Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  11. Enzyme and Microbial Development | Bioenergy | NREL

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Enzyme and Microbial Development NREL's enzyme and microbial development researchers improve the performance of enzymes and microbes for fuels and chemicals production from a variety of biomass feedstocks. In the process, we employ physical chemistry, biochemistry, computational modeling, and synthetic biology. Featured Publications Revealing Nature's Cellulase Diversity: The hyperactive CelA from Caldicellulosiruptor bescii, Science (2013) How Does Plant Cell Wall Nanoscale Architecture

  12. Targeted Enhancement of H2 and CO2 Uptake for Autotrophic Production of Biodiesel in the Lithoautotrophic Bacterium Ralsonia Eutropha

    SciTech Connect (OSTI)

    Eckert, C. A.; Sullivan, R.; Johnson, C.; Yu, J.; Maness, P. C.

    2013-01-01

    CO2 and H2 are promising feedstocks for production of valuable biocompounds. Ralstonia eutropha utilizes these feedstocks to generate energy (ATP) and reductant (NAD(P)H) via oxidation of H2 by a membrane-bound (MBH) and a soluble hydrogenase (SH) for CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle. Increased expression of the enzyme that fixes CO2 (RubisCO) resulted in 6-fold activity improvement in vitro, while increased expression of the MBH operon or the SH operon plus MBH operon maturation factors necessary for activity resulted in a 10-fold enhancement. Current research involves genetic manipulation of two endogenous cbb operons for increased expression, analysis of expression and activity of CBB/MBH/SH, cofactor ratios, and downstream products during autotrophic growth in control versus enhanced strains, and development of strategies for long-term, optimal overexpression. These studies will improve our understanding of autotrophic metabolism and provide a chassis strain for autotrophic production of biodiesel and other valuable carbon biocompounds.

  13. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the BacteriumNovosphingobium aromaticivorans

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; Meng, Da; Zhao, Rui; Toli?, Nikola; Cao, Li; Shukla, Anil; Monroe, Matthew E.; Moore, Ronald J.; et al

    2013-01-01

    The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome ofNovosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMsmoreincluding signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.less

  14. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; Meng, Da; Zhao, Rui; Tolić, Nikola; Cao, Li; Shukla, Anil; Monroe, Matthew E.; Moore, Ronald J.; et al

    2013-01-01

    The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans . Our top-down analysis provided the confident identification of 55 proteins in the periplasm andmore » characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.« less

  15. Laboratory Directed Research & Development program. Annual report to the Department of Energy

    SciTech Connect (OSTI)

    Ogeka, G.J.; Romano, A.J.

    1995-12-01

    This report briefly discusses the following projects coordinated at Brookhaven National Laboratory: investigation of the utility of max-entropy methods for the analysis of powder diffraction data; analysis of structures and interactions of nucleic acids and proteins by small angle x-ray diffraction; relaxographic MRI and functional MRI; very low temperature infra-red laser absorption as a potential analytical tool; state-resolved measurements of H{sub 2} photodesorption: development of laser probes of H{sub 2} for in-situ accelerator measurements; Siberian snake prototype development for RHIC; synthesis and characterization of novel microporous solids; ozone depletion, chemistry and physics of stratospheric aerosols; understanding the molecular basis for the synthesis of plant fatty acids possessing unusual double bond positions; structure determination of outer surface proteins of the Lyme disease spirochete; low mass, low-cost multi-wire proportional chambers for muon systems of collider experiments; theory of self-organized criticality; development of the PCR-SSCP technique for the detection, at the single cell level, of specific genetic changes; feasibility of SPECT in imaging of F-18 FDG accumulation in tumors; visible free electron laser oscillator experiment; study of possible 2 + 2 TeV muon-muon collider; ultraviolet FEL R & D; precision machining using hard x-rays; new directions in in-vivo enzyme mapping: catechol-O-methyltransferase; proposal to develop a high rate muon polarimeter; development of intense, tunable 20-femtosecond laser systems; use of extreme thermophilic bacterium thermatoga maritima as a source of ribosomal components and translation factors for structural studies; and biochemical and structural studies of Chaperon proteins from thermophilic bacteria and other experiments.

  16. Functional Heterologous Expression of an Engineered Full Length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

    SciTech Connect (OSTI)

    Currie, Devin; Herring, Christopher; Guss, Adam M; Olson, Daniel G.; Hogsett, David; Lynd, Lee R

    2013-01-01

    BACKGROUND: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. RESULTS: We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a cipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. CONCLUSION: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.

  17. System and method for introduction and stabilization of genes in Thermus sp.

    DOE Patents [OSTI]

    Kayser, Kevin J.; Park, Ho-Shin; Kilbane, II, John J.

    2005-03-01

    A method for introducing and stabilizing heterologous and recombinant genes in a thermophilic host in which a characteristic gene defining a detectable host characteristic is inactivated or deleted from the thermophilic host, resulting in a modified thermophilic host expressing an absence of the detectable host characteristic. A DNA fragment of interest is inserted into the modified thermophilic host together with an intact characteristic gene, whereby the detectable host characteristic is restored to the thermophilic host, thereby enabling detection and confirmation of successful transformation using plasmid vectors and integration of the DNA fragment into the chromosome of the thermophilic host.

  18. Liquid Fuel from Heat-Loving Microorganisms: H2-Dependent Conversion of CO2 to Liquid Electrofuels by Extremely Thermophilic Archaea

    SciTech Connect (OSTI)

    2010-07-01

    Electrofuels Project: NC State is working with the University of Georgia to create Electrofuels from primitive organisms called extremophiles that evolved before photosynthetic organisms and live in extreme, hot water environments with temperatures ranging from 167-212 degrees Fahrenheit The team is genetically engineering these microorganisms so they can use hydrogen to turn carbon dioxide directly into alcohol-based fuels. High temperatures are required to distill the biofuels from the water where the organisms live, but the heat-tolerant organisms will continue to thrive even as the biofuels are being distilledmaking the fuel-production process more efficient. The microorganisms dont require light, so they can be grown anywhereinside a dark reactor or even in an underground facility.

  19. Comparison of different procedures to stabilize biogas formation after process failure in a thermophilic waste digestion system: Influence of aggregate formation on process stability

    SciTech Connect (OSTI)

    Kleyboecker, A.; Liebrich, M.; Kasina, M.; Kraume, M.; Wittmaier, M.; Wuerdemann, H.

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Mechanism of process recovery with calcium oxide. Black-Right-Pointing-Pointer Formation of insoluble calcium salts with long chain fatty acids and phosphate. Black-Right-Pointing-Pointer Adsorption of VFAs by the precipitates resulting in the formation of aggregates. Black-Right-Pointing-Pointer Acid uptake and phosphate release by the phosphate-accumulating organisms. Black-Right-Pointing-Pointer Microbial degradation of volatile fatty acids in the aggregates. - Abstract: Following a process failure in a full-scale biogas reactor, different counter measures were undertaken to stabilize the process of biogas formation, including the reduction of the organic loading rate, the addition of sodium hydroxide (NaOH), and the introduction of calcium oxide (CaO). Corresponding to the results of the process recovery in the full-scale digester, laboratory experiments showed that CaO was more capable of stabilizing the process than NaOH. While both additives were able to raise the pH to a neutral milieu (pH > 7.0), the formation of aggregates was observed particularly when CaO was used as the additive. Scanning electron microscopy investigations revealed calcium phosphate compounds in the core of the aggregates. Phosphate seemed to be released by phosphorus-accumulating organisms, when volatile fatty acids accumulated. The calcium, which was charged by the CaO addition, formed insoluble salts with long chain fatty acids, and caused the precipitation of calcium phosphate compounds. These aggregates were surrounded by a white layer of carbon rich organic matter, probably consisting of volatile fatty acids. Thus, during the process recovery with CaO, the decrease in the amount of accumulated acids in the liquid phase was likely enabled by (1) the formation of insoluble calcium salts with long chain fatty acids, (2) the adsorption of volatile fatty acids by the precipitates, (3) the acid uptake by phosphorus-accumulating organisms and (4) the degradation of volatile fatty acids in the aggregates. Furthermore, this mechanism enabled a stable process performance after re-activation of biogas production. In contrast, during the counter measure with NaOH aggregate formation was only minor resulting in a rapid process failure subsequent the increase of the organic loading rate.

  20. Final Report on Development of Thermoanaerobacterium saccharolyticum for the conversion of lignocellulose to ethanol

    SciTech Connect (OSTI)

    Herring, Christopher D.; Kenealy, William R.; Shaw, A. Joe; Raman, Babu; Tschaplinski, Timothy J.; Brown, Steven D.; Davison, Brian H.; Covalla, Sean F.; Sillers, W. Ryan; Xu, Haowen; Tsakraklides, Vasiliki; Hogsett, David A.

    2012-01-24

    This project addressed the need for economical technology for the conversion of lignocellulosic biomass to fuels, specifically the conversion of pretreated hardwood to ethanol. The technology developed is a set of strains of the bacterium Thermoanaerobacterium saccharolyticum and an associated fermentation process for pretreated hardwood. Tools for genetic engineering and analysis of the organism were developed, including a markerless mutation method, a complete genome sequence and a set of gene expression profiles that show the activity of its genes under a variety of conditions relevant to lignocellulose conversion. Improved strains were generated by selection and genetic engineering to be able to produce higher amounts of ethanol (up to 70 g/L) and to be able to better tolerate inhibitory compounds from pretreated hardwood. Analysis of these strains has generated useful insight into the genetic basis for desired properties of biofuel producing organisms. Fermentation conditions were tested and optimized to achieve ethanol production targets established in the original project proposal. The approach proposed was to add cellulase enzymes to the fermentation, a method called Simultaneous Saccharification and Fermentation (SSF). We had reason to think SSF would be an efficient approach because the optimal temperature and pH for the enzymes and bacterium are very close. Unfortunately, we discovered that commercially available cellulases are inactivated in thermophilic SSF by a combination of low redox potential and ethanol. Despite this, progress was made against the fermentation targets using bacterial cellulases. Thermoanaerobacterium saccharolyticum may still prove to be a commercially viable technology should cellulase enzyme issues be addressed. Moreover, the organism was demonstrated to produce ethanol at approximately theoretical yield from oligomeric hemicellulose extracts, an ability that may prove to be uniquely valuable in pretreatment configurations in which cellulose and hemicellulose are separated.

  1. Microbiology and physiology of anaerobic fermentations of cellulose. Progress report

    SciTech Connect (OSTI)

    Peck, H.D. Jr.; Ljungdahl, L.G.; Mortenson, L.E.; Wiegel, J.K.W.

    1994-11-01

    This project studies the biochemistry and physiology of four major groups (primary, secondary, ancillary and methane bacteria) of anaerobic bacteria, that are involved in the conversion of cellulose to methane or chemical feedstocks. The primary bacterium, Clostridium thermocellum, has a cellulolytic enzyme system capable of hydrolyzing crystalline cellulose and consists of polypeptide complexes attached to the substrate cellulose with the aid of a low molecular yellow affinity substance (YAS) produced by the bacterium in the presence of cellulose. Properties of the complexes and YAS are studied. Aspects of metabolism are being studied which appear to be relevant for the interactions on consortia and their bioenergetics, particularly related to hydrogen, formate, CO, and CO{sub 2}. The roles of metals in the activation of H{sub 2} are being investigated, and genes for the hydrogenases cloned and sequenced to established structural relationships among the hydrogenases. The goals are to understand the roles and regulation of hydrogenases in interspecies H{sub 2} transfer, H{sub 2} cycling and the generation of a proton gradient. The structures of the metal clusters and their role in the metabolism of formate will be investigated with the goal of understanding the function of formate in the total synthesis of acetate from CO{sub 2} and its role in the bioenergetics of these microorganisms. Additionally, the enzyme studies will be performed using thermophiles and also the isolation of some new pertinent species. The project will also include research on the mechanism of extreme thermophily (growth over 70{degrees}) in bacteria that grow over a temperature span of 40{degrees}C or more. These bacteria exhibit a biphasic growth response to temperature and preliminary evidence suggests that the phenomenon is due to the expression of a new set of enzymes. These initial observations will be extended employing techniques of molecular biology.

  2. Transgenic Plants Lower the Costs of Cellulosic Biofuels (Fact Sheet)

    SciTech Connect (OSTI)

    Not Available

    2011-11-01

    A new transgenic maize was observed to be less recalcitrant than wild-type biomass, as manifested through lower severity requirements to achieve comparable levels of conversion. Expression of a single gene derived from bacteria in plants has resulted in transgenic plants that are easier and cheaper to convert into biofuels. Part of the high production cost of cellulosic biofuels is the relatively poor accessibility of substrates to enzymes due to the strong associations between plant cell wall components. This biomass recalcitrance makes costly thermochemical pretreatment necessary. Scientists at the National Renewable Energy Laboratory (NREL) have created transgenic maize expressing an active glycosyl hydrolase enzyme, E1 endoglucanase, originally isolated from a thermophilic bacterium, Acidothermus cellulolyticus. This engineered feedstock was observed to be less recalcitrant than wild-type biomass when subjected to reduced severity pretreatments and post-pretreatment enzymatic hydrolysis. This reduction in recalcitrance was manifested through lower severity requirements to achieve comparable levels of conversion of wild-type biomass. The improvements observed are significant enough to positively affect the economics of the conversion process through decreased capital construction costs and decreased degradation products and inhibitor formation.

  3. Deletion of the Cel48S cellulase from Clostridium thermocellum

    SciTech Connect (OSTI)

    Olson, Daniel G; Tripathi, Shital A.; Giannone, Richard J; Lo, Jonathan; Caiazza, Nicky; Hogsett, David A; Hettich, Robert {Bob} L; Guss, Adam M; Dubrovsky, Genia; Lynd, Lee R

    2010-01-01

    Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, SS, and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced by the S mutant strain was purified by affinity digestion, characterized enzymatically, and found to have a 35% lower specific activity on Avicel. The composition of the purified cellulosome was analyzed by tandem mass spectrometry with APEX quantification and no significant changes in abundance were observed in any of the major (>1% of cellulosomal protein) enzymatic subunits. Although most cellulolytic bacteria have one family 48 cellulase, C. thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout was essentially the same as that of the Cel48S single knockout. Our results indicate that solubilization of crystalline cellulose by C. thermocellum can proceed to completion without expression of a family 48 cellulase.

  4. Relationship between the fine structure of native cellulose and cellulose degradability by the cellulase complexes of Trichoderma reesei and Clostridium thermocellum

    SciTech Connect (OSTI)

    Weimer, P.J.; Weston, W.M.

    1985-11-01

    The initial rate of hydrolysis of six commercially available native (type 1) celluloses was determined for the crude cellulase complexes of the thermophilic anaerobic bacterium C. thermocellum and the mesophilic fungus T. reesei. These rates were then compared with certain physical features of the substrates in an attempt to determine the role of cellulose structure in its degradability. Within the substrate series tested, the Clostridium system showed a greater relative range in rate of enzymatic hydrolysis than did the Trichoderma system. Average correlation coefficients for the kinetic rates from bacterial and fungal cellulases, respectively, and the following physical parameters were obtained: relative crystallinity index (RCI) from acid hydrolysis, -0.61 and -0.85; RCI from x-ray diffraction, -0.75 and -0.89; accessibility to formylation at 4 degrees C, +0.49 and +0.60; nonaccessibility to formylation at 65 degrees, -0.40 and - 0.73; fiber saturation point, +0.83 and +0.85. Kinetic and pore volume distribution data suggest that the rate-limiting components of both the bacterial and fungal cellulase systems are of similar size, approximately 43 Angstroms along one axis. 32 references.

  5. Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray

    SciTech Connect (OSTI)

    Brown, Steven David; Raman, Babu; McKeown, Catherine K; Kale, Shubhangi P; He, Zhili; Mielenz, Jonathan R

    2007-04-01

    Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

  6. Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca

    SciTech Connect (OSTI)

    Burchhardt, G.; Ingram, L.O. )

    1992-04-01

    A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestability of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.

  7. Impact of Pretreated Switchgrass and Biomass Carbohydrates on Clostridium thermocellum 27405 Cellulosome Composition- a Quantitative Proteomic Analysis

    SciTech Connect (OSTI)

    Raman, Babu; Pan, Chongle; Hurst, Gregory {Greg} B; Rodriguez, Jr., Miguel; McKeown, Catherine K; Lankford, Patricia K; Samatova, Nagiza F; Mielenz, Jonathan R

    2009-01-01

    The anaerobic thermophilic bacterium Clostridium thermocellum is a cellulolytic organism capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolic products. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed the cellulosomes. In this study, we used quantitative proteomics (multidimensional LC-MS/MS and 15N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to 15N labeled cellulosome samples isolated from crystalline cellulose grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 15 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline. Overall, the results suggest a coordinated substrate-specific regulation of cellulosomal composition in C. thermocellum.

  8. Genome Sequence of Thermotoga sp Strain RQ2, a Hyperthermophilic Bacterium Isolated from a Geothermally Heated Region of the Seafloor near Ribeira Quente, the Azores

    SciTech Connect (OSTI)

    Swithers, Kristen S; DiPippo, Jonathan L; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Woyke, Tanja; Pitluck, Sam; Pennacchio, Len; Nolan, Matt; Mikhailova, Natalia; Lykidis, A; Land, Miriam L; Stetter, Karl O; Nelson, Karen E; Gogarten, Peter; Noll, Kenneth M

    2011-01-01

    Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.

  9. CO2 exposure at pressure impacts metabolism and stress responses in the model sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough

    SciTech Connect (OSTI)

    Wilkins, Michael J.; Hoyt, David W.; Marshall, Matthew J.; Alderson, Paul A.; Plymale, Andrew E.; Markillie, Lye Meng; Tucker, Abigail E.; Walter, Eric D.; Linggi, Bryan E.; Dohnalkova, Alice; Taylor, Ronald C.

    2014-09-01

    Geologic carbon dioxide (CO2) sequestration drives physical and geochemical changes in deep subsurface environments that impact indigenous microbial activities. The combined effects of pressurized CO2 on a model sulfate-reducing microorganism, Desulfovibrio vulgaris, have been assessed using a suite of genomic and kinetic measurements. Novel high-pressure NMR time-series measurements using 13C-lactate were used to track D. vulgaris metabolism. We identified cessation of respiration at CO2 pressures of 10 bar, 25 bar, 50 bar, and 80 bar. Concurrent experiments using N2 as the pressurizing phase had no negative effect on microbial respiration, as inferred from reduction of sulfate to sulfide. Complementary pressurized batch incubations and fluorescence microscopy measurements supported NMR observations, and indicated that non-respiring cells were mostly viable at 50 bar CO2 for at least four hours, and at 80 bar CO2 for two hours. The fraction of dead cells increased rapidly after four hours at 80 bar CO2. Transcriptomic (RNA-Seq) measurements on mRNA transcripts from CO2-incubated biomass indicated that cells up-regulated the production of certain amino acids (leucine, isoleucine) following CO2 exposure at elevated pressures, likely as part of a general stress response. Evidence for other poorly understood stress responses were also identified within RNA-Seq data, suggesting that while pressurized CO2 severely limits the growth and respiration of D. vulgaris cells, biomass retains intact cell membranes at pressures up to 80 bar CO2. Together, these data show that geologic sequestration of CO2 may have significant impacts on rates of sulfate reduction in many deep subsurface environments where this metabolism is a key respiratory process.

  10. Complete genome sequence of Anaeromyxobacter sp. Fw109-5, an Anaerobic, Metal-Reducing Bacterium Isolated from a Contaminated Subsurface Environment

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Hwang, C.; Copeland, A.; Lucas, Susan; Lapidus, Alla; Barry, Kerrie W.; Glavina del Rio, T.; Dalin, Eileen; Tice, Hope; Pitluck, S.; Sims, David R.; et al

    2015-01-22

    We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacterium’s genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation.

  11. Non-contiguous finished genome sequence and contextual data of the filamentous soil bacterium Ktedonobacter racemifer type strain (SOSP1-21T)

    SciTech Connect (OSTI)

    Chang, Yun-Juan [ORNL; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Chertkov, Olga [Los Alamos National Laboratory (LANL); Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Brettin, Thomas S [ORNL; Fiebig, Anne [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Abt, Birte [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute

    2011-01-01

    Ktedonobacter racemifer corrig. Cavaletti et al. 2007 is the type species of the genus Ktedo- nobacter, which in turn is the type genus of the family Ktedonobacteraceae, the type family of the order Ktedonobacterales within the class Ktedonobacteria in the phylum Chloroflexi . Although K. racemifer shares some morphological features with the actinobacteria, it is of special interest because it was the first cultivated representative of a deep branching unclassi- fied lineage of otherwise uncultivated environmental phylotypes tentatively located within the phylum Chloroflexi . The aerobic, filamentous, non-motile, spore-forming Gram-positive heterotroph was isolated from soil in Italy. The 13,661,586 bp long non-contiguous finished genome consists of ten contigs and is the first reported genome sequence from a member of the class Ktedonobacteria. With its 11,453 protein-coding and 87 RNA genes, it is the largest prokaryotic genome reported so far. It comprises a large number of over-represented COGs, particularly genes associated with transposons, causing the genetic redundancy within the genome being considerably larger than expected by chance. This work is a part of the Ge- nomic Encyclopedia of Bacteria and Archaea project.

  12. Binding and Direct Electrochemistry of OmcA, an Outer-Membrane Cytochrome from an Iron Reducing Bacterium, with Oxide Electrodes: A Candidate Biofuel Cell System

    SciTech Connect (OSTI)

    Eggleston, Carrick M.; Voros, Janos; Shi, Liang; Lower, Brian H.; Droubay, Timothy C.; Colberg, Patricia J.

    2008-02-15

    Dissimilatory iron-reducing bacteria transfer electrons to solid ferric respiratory electron acceptors. Outer-membrane cytochromes expressed by these organisms are of interest in both microbial fuel cells and biofuel cells. We use optical waveguide lightmode spectroscopy (OWLS) to show that OmcA, an 85 kDa decaheme outer-membrane c-type cytochrome from Shewanella oneidensis MR-1, adsorbs to isostructural Al2O3 and Fe2O3 in similar amounts. Adsorption is ionic-strength and pH dependent (peak adsorption at pH 6.57.0). The thickness of the OmcA layer on Al2O3 at pH 7.0 [5.8 1.1 (2r) nm] from OWLS is similar, within error, to that observed using atomic force microscopy (4.8 2 nm). The highest adsorption density observed was 334 ng cm 2 (2.4 1012 molecules cm 2), corresponding to a monolayer or 9.9 nm diameter spheres or submonolayer coverage by smaller molecules. Direct electrochemistry of OmcA on Fe2O3 electrodes was observed using cyclic voltammetry, with cathodic peak potentials of 380 to 320 mV versus Ag/AgCl. Variations in the cathodic peak positions are speculatively attributed to redox-linked conformation change or changes in molecular orientation. OmcA can exchange electrons with ITO electrodes at higher current densities than with Fe2O3. Overall, OmcA can bind to and exchange electrons with several oxides, and thus its utility in fuel cells is not restricted to Fe2O3.

  13. Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic

    Office of Scientific and Technical Information (OSTI)

    Thermophile Alvinella pompejana (Journal Article) | SciTech Connect SciTech Connect Search Results Journal Article: Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana Citation Details In-Document Search Title: Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana Human DNA polymerase η (HsPol η ) plays an important role in translesion synthesis (TLS), which allows for replication past DNA

  14. TMO Renewables Ltd formerly TMO Biotec Ltd | Open Energy Information

    Open Energy Info (EERE)

    Energy Product: Startup working on the use of thermophile (high-temperature) fermentation to replace yeast-based fermentation for the production of renewable ethanol, and...

  15. Microsoft Word - Mills CV March 2015.docx

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    ... Mills, C.S. Romanek and J. Wiegel (2005) Thermosediminibacter oceani and Thermosedminibacter litoriperuensis, new anaerobic thermophilic bacteria isolated from the Peru margin. ...

  16. Biocaldol | Open Energy Information

    Open Energy Info (EERE)

    Biocaldol Jump to: navigation, search Name: Biocaldol Place: London, England, United Kingdom Zip: NW1 0NH Sector: Biomass Product: Biocaldol uses thermophilic microorganisms to...

  17. Search for: All records | SciTech Connect

    Office of Scientific and Technical Information (OSTI)

    ... provided. July 2009 Copy of Synthetic Biology of Novel Thermophilic Bacteria for ... July 2009 Molecular Simulation of Protein Aggregation Bratko, Dusan ; Cellmer, Troy ; ...

  18. New generation NMR bioreactor coupled with high-resolution NMR spectroscopy leads to novel discoveries in Moorella thermoaceticum metabolic profiles

    SciTech Connect (OSTI)

    Xue, Junfeng; Isern, Nancy G.; Ewing, R James; Liyu, Andrey V.; Sears, Jesse A.; Knapp, Harlan; Iversen, Jens; Sisk, Daniel R.; Ahring, Birgitte K.; Majors, Paul D.

    2014-06-20

    An in-situ nuclear magnetic resonance (NMR) bioreactor was developed and employed to monitor microbial metabolism under batch-growth conditions in real time. We selected Moorella thermoacetica ATCC 49707 as a test case. M. thermoacetica (formerly Clostridium thermoaceticum) is a strictly anaerobic, thermophilic, acetogenic, gram-positive bacterium with potential for industrial production of chemicals. The metabolic profiles of M. thermoacetica were characterized during growth in batch mode on xylose (a component of lignocellulosic biomass) using the new generation NMR bioreactor in combination with high-resolution, high sensitivity NMR (HR-NMR) spectroscopy. In-situ NMR measurements were performed using water-suppressed H-1 NMR spectroscopy at an NMR frequency of 500 MHz, and aliquots of the bioreactor contents were taken for 600 MHz HR-NMR spectroscopy at specific intervals to confirm metabolite identifications and expand metabolite coverage. M. thermoacetica demonstrated the metabolic potential to produce formate, ethanol and methanol from xylose, in addition to its known capability of producing acetic acid. Real-time monitoring of bioreactor conditions showed a temporary pH decrease, with a concomitant increase in formic acid during exponential growth. Fermentation experiments performed outside of the magnet showed that the strong magnetic field employed for NMR detection did not significantly affect cell metabolism. Use of the in-situ NMR bioreactor facilitated monitoring of the fermentation process in real time, enabling identification of intermediate and end-point metabolites and their correlation with pH and biomass produced during culture growth. Real-time monitoring of culture metabolism using the NMR bioreactor in combination with the HR-NMR spectroscopy will allow optimization of the metabolism of microorganisms producing valuable bioproducts.

  19. Berkeley Lab - Materials Sciences Division

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    How to Train Your Bacterium Peidong Yang, a chemist with Berkeley Lab's Materials Sciences Division, and his researchers are using the bacterium Moorella thermoacetica to perform...

  20. Photobiology Research Laboratory (Fact Sheet), NREL (National...

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    reinhardtii (green alga) - Synechocystis 6803 (cyanobacterium) - Clostridium thermocellum (fermentative bacterium) - Ralstonia eutropha (chemolithoautotroph) * Purification ...

  1. Recoding of the stop codon UGA to glycine by a BD1-5/SN-2 bacterium and niche partitioning between Alpha- and Gammaproteobacteria in a tidal sediment microbial community naturally selected in a laboratory chemostat

    SciTech Connect (OSTI)

    Hanke, Anna; Hamann, Emmo; Sharma, Ritin; Geelhoed, Jeanine; Hargesheimer, Theresa; Kraft, Beate; Meyer, Volker; Lenk, Sabine; Osmers, Harald; Wu, Rong; Makinwa, Kofi; Hettich, Robert {Bob} L; Banfield, Jillian F.; Tegetmeyer, Halina; Strouss, Marc

    2014-01-01

    Sandy coastal sediments are global hot spots for microbial mineralization of organic matter and denitrification. These sediments are characterized by advective pore water flow, tidal cycling and an active and complex microbial community. Metagenomic sequencing of microbial communities sampled from such sediments showed that potential sulfuroxidizing Gammaproteobacteria and members of the enigmaticBD1-5/ SN-2 candidatephylumwereabundantinsitu (>10% and 2% respectively). By mimicking the dynamic oxic/anoxic environmental conditions of the sedimentin a laboratory chemostat, a simplified microbial community was selected from the more complex inoculum. Metagenomics, proteomics and fluorescenceinsituhybridization showed that this simplified community contained both a potential sulfuroxidizing Gamma proteobacteria (at 24 2% abundance) and a member of the BD1-5 / SN-2candidatephylum (at 7 6%abundance). Despite the abundant supply of organic substrates to the chemostat, proteomic analysis suggested that the selected gamma proteobacterium grew partially auto trophically and performed hydrogen/formate oxidation. The enrichment of a member of the BD1-5/SN-2candidatephylum enabled, for the first time, direct microscopic observation by fluorescent insitu hybridization and the experimental validation of the previously predicted translation of the stop codon UGA into glycine.

  2. High-Quality Draft Genome Sequence of Desulfovibrio carbinoliphilus FW-101-2B, an Organic Acid-Oxidizing Sulfate-Reducing Bacterium Isolated from Uranium(VI)-Contaminated Groundwater

    SciTech Connect (OSTI)

    Ramsay, Bradley D.; Hwang, Chiachi; Woo, Hannah L.; Carroll, Sue L.; Lucas, Susan; Han, James; Lapidus, Alla L.; Cheng, Jan-Fang; Goodwin, Lynne A.; Pitluck, Samuel; Peters, Lin; Chertkov, Olga; Held, Brittany; Detter, John C.; Han, Cliff S.; Tapia, Roxanne; Land, Miriam L.; Hauser, Loren J.; Kyrpides, Nikos C.; Ivanova, Natalia N.; Mikhailova, Natalia; Pagani, Loanna; Woyke, Tanja; Arkin, Adam P.; Dehal, Paramvir; Chivian, Dylan; Criddle, Craig S.; Wu, Weimin; Chakraborty, Romy; Hazen, Terry C.; Fields, Matthew W.

    2015-03-12

    Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing ?-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction.

  3. High-Quality Draft Genome Sequence of Desulfovibrio carbinoliphilus FW-101-2B, an Organic Acid-Oxidizing Sulfate-Reducing Bacterium Isolated from Uranium(VI)-Contaminated Groundwater

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Ramsay, Bradley D.; Hwang, Chiachi; Woo, Hannah L.; Carroll, Sue L.; Lucas, Susan; Han, James; Lapidus, Alla L.; Cheng, Jan-Fang; Goodwin, Lynne A.; Pitluck, Samuel; et al

    2015-03-12

    Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing δ-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction.

  4. Bacillus MGA3 aspartokinase II gene

    DOE Patents [OSTI]

    Schendel, Frederick J.; Flickinger, Michael C.

    1993-01-01

    The present invention provides the isolated DNA sequence encoding the .alpha.B dimer subunit of the lysine-sensitive aspartokinase II isozyme from the thermophilic methylotrophic Bacillus sp. MGA3.

  5. Enviro Control Ltd ECL | Open Energy Information

    Open Energy Info (EERE)

    Ltd ECL Jump to: navigation, search Name: Enviro-Control Ltd (ECL) Place: Cardiff, United Kingdom Zip: CF2 7HP Product: A developer of proprietary thermophilic anaerobic digestion...

  6. Yannick J. Bomble, Ph.D. | Bioenergy | NREL

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Yannick J. Bomble Yannick J. Bomble, Ph.D. Senior Research Scientist Yannick.Bomble@nrel.gov | 303-384-7729 Research Interests Development of thermophilic bacteria for improved biomass deconstruction Biomass degrading mechanisms of thermophilic bacteria Metabolic modeling and pathway engineering in microbes for upgrading sugar to fuels and high value chemicals Metabolic enzyme characterization, modeling, and engineering (thermostability, cofactor specificity, binding, rate limiting step)

  7. The winds of (evolutionary) change: Breathing new life into microbiology

    SciTech Connect (OSTI)

    Olsen, G.J.; Woese, C.R.; Overbeek, R.A.

    1996-03-01

    To date, over 1500 prokaryotes have been characterized by small subunit rRNA sequencing and molecular phylogeny has had an equally profound effect on our understanding of relationship among eukaryotic microorganisms. The universal phylogenetic tree readily shows however how artificial the strong distinction between the eukaryote and prokaryotes has become. The split between the Archaea and the Bacteria is now recognized as the primary phylogenetic division and that the Eucarya have branched from the same side of the tree as the Archaea. Both prokaryotic domains would seem to be of thermophilic origin suggesting that life arose in a very warm environment. Among the Archaea, all of the Crenarchaeota cultured to date are thermophiles, and the deepest euryarchaeal branchings are represented exclusively by thermophiles. Among the Bacteria, the deepest known branchings are again represented exclusively by thermophiles, and thermophilia is widely scattered throughout the domain. The Archaea comprise a small number of quite disparate phenotypes that grow in unusual niches. All are obligate or facultative anaerobes. All cultured crenarchaeotes are thermophilic, some even growing optimally above the normal boiling temperature of water. The Archaeoglobales are sulfate reducers growing at high temperatures. The extreme halophiles grow only in highly saline environments. The methanogens are confined to a variety of anaerobic niches, often thermophilic. The Bacteria, on the other hand, are notable as being the source of life`s photosynthetic capacity. Five kingdoms of bacteria contain photosynthetic species; and each of the five manifests a distinct type of (chlorophyll-based) photosynthesis.

  8. The Winds of (Evolutionary) Change: Breathing New Life into Microbiology

    DOE R&D Accomplishments [OSTI]

    Olsen, G. J.; Woese, C. R; Overbeek, R. A.

    1996-03-01

    To date, over 1500 prokaryotes have been characterized by small subunit rRNA sequencing and molecular phylogeny has had an equally profound effect on our understanding of relationship among eukaryotic microorganisms. The universal phylogenetic tree readily shows however how artificial the strong distinction between the eukaryote and prokaryotes has become. The split between the Archaea and the Bacteria is now recognized as the primary phylogenetic division and that the Eucarya have branched from the same side of the tree as the Archaea. Both prokaryotic domains would seem to be of thermophilic origin suggesting that life arose in a very warm environment. Among the Archaea, all of the Crenarchaeota cultured to date are thermophiles, and the deepest euryarchaeal branchings are represented exclusively by thermophiles. Among the Bacteria, the deepest known branchings are again represented exclusively by thermophiles, and thermophilia is widely scattered throughout the domain. The Archaea comprise a small number of quite disparate phenotypes that grow in unusual niches. All are obligate or facultative anaerobes. All cultured crenarchaeotes are thermophilic, some even growing optimally above the normal boiling temperature of water. The Archaeoglobales are sulfate reducers growing at high temperatures. The extreme halophiles grow only in highly saline environments. The methanogens are confined to a variety of anaerobic niches, often thermophilic. The Bacteria, on the other hand, are notable as being the source of life`s photosynthetic capacity. Five kingdoms of bacteria contain photosynthetic species; and each of the five manifests a distinct type of (chlorophyll-based) photosynthesis.

  9. Ethanologenic bacteria with increased resistance to furfural

    DOE Patents [OSTI]

    Miller, Elliot Norman; Jarboe, Laura R.; Yomano, Lorraine P.; York, Sean W.; Shanmugam, Keelnatham; Ingram, Lonnie O'Neal

    2015-10-06

    The invention relates to bacterium that have increased resistance to furfural and methods of preparation. The invention also relates to methods of producing ethanol using the bacterium and corresponding kits.

  10. Berkeley Lab

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Berkeley Lab Scientists Teach Bacterium a New Trick for Artificial Photosynthesis http:www.lbl.gov20160108berkeley-lab-scientists-teach-bacterium-a-new-trick-for-artificial-p...

  11. Search for: All records | SciTech Connect

    Office of Scientific and Technical Information (OSTI)

    ... The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, ... D. baculatum strain XT is a Gram-negative, motile, sulfate-reducing bacterium isolated ...

  12. Dispersant solutions for dispersing hydrocarbons

    DOE Patents [OSTI]

    Tyndall, R.L.

    1997-03-11

    A dispersant solution includes a hydrocarbon dispersing solution derived from a bacterium from ATCC 75527, ATCC 75529, or ATCC 55638.

  13. Dispersant solutions for dispersing hydrocarbons

    DOE Patents [OSTI]

    Tyndall, Richard L. (Clinton, TN)

    1997-01-01

    A dispersant solution includes a hydrocarbon dispersing solution derived from a bacterium from ATCC 75527, ATCC 75529, or ATCC 55638.

  14. Modified cyanobacteria

    SciTech Connect (OSTI)

    Vermaas, Willem F J.

    2014-06-17

    Disclosed is a modified photoautotrophic bacterium comprising genes of interest that are modified in terms of their expression and/or coding region sequence, wherein modification of the genes of interest increases production of a desired product in the bacterium relative to the amount of the desired product production in a photoautotrophic bacterium that is not modified with respect to the genes of interest.

  15. Isolation of Clostridium thermocellum auxotrophs

    SciTech Connect (OSTI)

    Mendez, B.S.; Gomez, R.F.

    1982-02-01

    The conversion of biomass of fuels and chemical feedstocks by microbial fermentation offers the potential of solving two of today's important problems: waste accumulation and exhaustion of fossil fuels. Microorganisms with the capabilities of converting biomass components such as cellulos and hemicellulose to chemicals and fuels in a single step are of particular interest. One such microorganism is Clostridium thermocellum, a thermophilic anaerobe which degrades cellulose to ethanol and organic acids. For efficient industrial use, the cellulolytic capacity of this strain must be improved by genetic means. Spontaneous and UV irradiation-induced auxotrophic mutants of Clostridium thermocellum, an anaerobic cellulolytic thermophile, were isolated after penicillin enrichment in a chemically defined medium.

  16. IMPACTS OF BIOFILM FORMATION ON CELLULOSE FERMENTATION

    SciTech Connect (OSTI)

    Leschine, Susan

    2009-10-31

    This project addressed four major areas of investigation: i) characterization of formation of Cellulomonas uda biofilms on cellulose; ii) characterization of Clostridium phytofermentans biofilm development; colonization of cellulose and its regulation; iii) characterization of Thermobifida fusca biofilm development; colonization of cellulose and its regulation; and iii) description of the architecture of mature C. uda, C. phytofermentans, and T. fusca biofilms. This research is aimed at advancing understanding of biofilm formation and other complex processes involved in the degradation of the abundant cellulosic biomass, and the biology of the microbes involved. Information obtained from these studies is invaluable in the development of practical applications, such as the single-step bioconversion of cellulose-containing residues to fuels and other bioproducts. Our results have clearly shown that cellulose-decomposing microbes rapidly colonize cellulose and form complex structures typical of biofilms. Furthermore, our observations suggest that, as cells multiply on nutritive surfaces during biofilms formation, dramatic cell morphological changes occur. We speculated that morphological changes, which involve a transition from rod-shaped cells to more rounded forms, might be more apparent in a filamentous microbe. In order to test this hypothesis, we included in our research a study of biofilm formation by T. fusca, a thermophilic cellulolytic actinomycete commonly found in compost. The cellulase system of T. fusca has been extensively detailed through the work of David Wilson and colleagues at Cornell, and also, genome sequence of a T. fusca strain has been determine by the DOE Joint Genome Institute. Thus, T. fusca is an excellent subject for studies of biofilm development and its potential impacts on cellulose degradation. We also completed a study of the chitinase system of C. uda. This work provided essential background information for understanding how C. uda colonizes and degrades insoluble substrates. Major accomplishments of the project include: • Development of media containing dialysis tubing (described by the manufacturer as “regenerated cellulose”) as sole carbon and energy source and a nutritive surface for the growth of cellulolytic bacteria, and development of various microscopic methods to image biofilms on dialysis tubing. • Demonstration that cultures of C. phytofermentans, an obligate anaerobe, C. uda, a facultative aerobe, and T. fusca, a filamentous aerobe, formed microbial communities on the surface of dialysis tubing, which possessed architectural features and functional characteristics typical of biofilms. • Demonstration that biofilm formation on the nutritive surface, cellulose, involves a complex developmental processes, including colonization of dialysis tubing, formation of cell clusters attached to the nutritive surface, cell morphological changes, formation of complex structures embedded in extracellular polymeric matrices, and dispersal of biofilm communities as the nutritive surface is degraded. • Determination of surface specificity and regulatory aspects of biofilm formation by C. phytofermentans, C. uda, and T. fusca. • Demonstration that biofilm formation by T. fusca forms an integral part of the life cycle of this filamentous cellulolytic bacterium, including studies on the role of mycelial pellet formation in the T. fusca life cycle and a comparison of mycelial pellets to surface-attached T. fusca biofilms. • Characterization of T. fusca biofilm EPS, including demonstration of a functional role for EPS constituents. • Correlation of T. fusca developmental life cycle and cellulase gene expression.

  17. Complete genome sequence of Kytococcus sedentarius type strain...

    Office of Scientific and Technical Information (OSTI)

    is a free-living, nonmotile, Gram-positive bacterium, originally isolated from a marine environment. Here we describe the features of this organism, together with the...

  18. Purple Bacteria Develops Its Own Form of Sunscreen | U.S. DOE...

    Office of Science (SC) Website

    Found that specific pigments in the light harvesting complex of a photosynthetic bacterium act primarily to protect the cell from damage by excess sunlight Significance and Impact ...

  19. 15570_JBEI_trifold_released2:Layout 1

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    fuel pro- duction systems in selected model microor- ganisms; the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, and the ex- tremophile archaeon Sulfolobus...

  20. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it...

  1. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    by the bacterium Clostridium botulinum, which can be ingested with contaminated food, enter the body through wounds, or (in the case of infants where there is little...

  2. Microsoft Word - Mn.doc

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    the identities of manganese oxides formed in sea water by the marine bacterium, Bacillus sp., strain SG-1. Both techniques provide information regarding the molecular-scale...

  3. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    The culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it...

  4. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it secretes...

  5. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    due to respiratory failure resulting from paralysis of the muscles used in breathing. The muscle paralysis is caused by the neurotoxin produced by the bacterium Clostridium...

  6. Fermilab Today - Safety Tip of the Week Archive

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    out a student loan in my name." September 28, 2009 Childhood vaccination for whooping cough wears off Whooping cough, caused by bordetella pertussis bacterium, was once thought...

  7. ORISE: Undergraduate Research Experiences - Ashley Cross

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    genetically engineer Clostridium thermocellum, a bacterium responsible for breaking ... "My main goals were to make C. thermocellum more tractable and to characterize a mutant ...

  8. Search for: All records | SciTech Connect

    Office of Scientific and Technical Information (OSTI)

    Research at ASU, focused on the biogenesis aspects, examined the biogeochemical bases for iron-sulfide production by Desulfovibrio vulgaris, a Gram-negative bacterium that is one ...

  9. Search for: All records | SciTech Connect

    Office of Scientific and Technical Information (OSTI)

    ... Research at ASU, focused on the biogenesis aspects, examined the biogeochemical bases for iron-sulfide production by Desulfovibrio vulgaris, a Gram-negative bacterium that is one ...

  10. Assessing the Role of Iron Sulfides in the Long Term Sequestration...

    Office of Scientific and Technical Information (OSTI)

    Research at ASU, focused on the biogenesis aspects, examined the biogeochemical bases for iron-sulfide production by Desulfovibrio vulgaris, a Gram-negative bacterium that is one ...

  11. Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution...

    Office of Scientific and Technical Information (OSTI)

    XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one ...

  12. CX-100328 Categorical Exclusion Determination

    Broader source: Energy.gov [DOE]

    Engineering Thermophiles to Produce Drop-In Biofuels from Syngas Award Number: DE-EE0007008 CX(s) Applied: A9, B3.6 Bioenergy Technologies Office Date: 08/12/2015 Location(s): CA Office(s): Golden Field Office

  13. Method for the detection of Salmonella enterica serovar Enteritidis

    DOE Patents [OSTI]

    Agron, Peter G.; Andersen, Gary L.; Walker, Richard L.

    2008-10-28

    Described herein is the identification of a novel Salmonella enterica serovar Enteritidis locus that serves as a marker for DNA-based identification of this bacterium. In addition, three primer pairs derived from this locus that may be used in a nucleotide detection method to detect the presence of the bacterium are also disclosed herein.

  14. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOE Patents [OSTI]

    McCready, Paula M. (Tracy, CA); Radnedge, Lyndsay (San Mateo, CA); Andersen, Gary L. (Berkeley, CA); Ott, Linda L. (Livermore, CA); Slezak, Thomas R. (Livermore, CA); Kuczmarski, Thomas A. (Livermore, CA); Vitalis, Elizabeth A (Livermore, CA)

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  15. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOE Patents [OSTI]

    McCready, Paula M. (Tracy, CA); Radnedge, Lyndsay (San Mateo, CA); Andersen, Gary L. (Berkeley, CA); Ott, Linda L. (Livermore, CA); Slezak, Thomas R. (Livermore, CA); Kuczmarski, Thomas A. (Livermore, CA); Vitalis, Elizabeth A (Livermore, CA)

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  16. Nucleotide sequences specific to Brucella and methods for the detection of Brucella

    DOE Patents [OSTI]

    McCready, Paula M. (Tracy, CA); Radnedge, Lyndsay (San Mateo, CA); Andersen, Gary L. (Berkeley, CA); Ott, Linda L. (Livermore, CA); Slezak, Thomas R. (Livermore, CA); Kuczmarski, Thomas A. (Livermore, CA)

    2009-02-24

    Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  17. Nucleotide sequences specific to Yersinia pestis and methods for the detection of Yersinia pestis

    DOE Patents [OSTI]

    McCready, Paula M. (Tracy, CA); Radnedge, Lyndsay (San Mateo, CA); Andersen, Gary L. (Berkeley, CA); Ott, Linda L. (Livermore, CA); Slezak, Thomas R. (Livermore, CA); Kuczmarski, Thomas A. (Livermore, CA); Motin, Vladinir L. (League City, TX)

    2009-02-24

    Nucleotide sequences specific to Yersinia pestis that serve as markers or signatures for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  18. Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

    DOE Patents [OSTI]

    Dees, H.C.

    1998-08-04

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials. 5 figs.

  19. Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

    SciTech Connect (OSTI)

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials.

  20. High temperature pre-digestion of corn stover biomass for improved product yields

    SciTech Connect (OSTI)

    Brunecky, Roman; Hobdey, Sarah E.; Taylor, Larry E.; Tao, Ling; Tucker, Melvin P.; Himmel, Michael E.; Decker, Stephen R.

    2014-12-03

    Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation. Results: We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions. In conclusion, Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.

  1. High temperature pre-digestion of corn stover biomass for improved product yields

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Brunecky, Roman; Hobdey, Sarah E.; Taylor, Larry E.; Tao, Ling; Tucker, Melvin P.; Himmel, Michael E.; Decker, Stephen R.

    2014-12-03

    Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation.more » Results: We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions. In conclusion, Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.« less

  2. Biochemistry and physiology of anaerobic bacteria

    SciTech Connect (OSTI)

    2000-05-18

    We welcome you to The Power of Anaerobes. This conference serves two purposes. One is to celebrate the life of Harry D. Peck, Jr.,who was born May 18, 1927 and would have celebrated his 73rd birthday at this conference. He died November 20, 1998. The second is to gather investigators to exchange views within the realm of anaerobic microbiology, an area in which tremendous progress has been seen during recent years. It is sufficient to mention discoveries of a new form of life (the archaea), hyper or extreme thermophiles, thermophilic alkaliphiles and anaerobic fungi. With these discoveries has come a new realization about physiological and metabolic properties of microorganisms, and this in turn has demonstrated their importance for the development, maintenance and sustenance of life on Earth.

  3. Improvements of biomass deconstruction enzymes

    SciTech Connect (OSTI)

    Sale, K. L.

    2012-03-01

    Sandia National Laboratories and DSM Innovation, Inc. collaborated on the investigation of the structure and function of cellulases from thermophilic fungi. Sandia's role was to use its expertise in protein structure determination and X-ray crystallography to solve the structure of these enzymes in their native state and in their substrate and product bound states. Sandia was also tasked to work with DSM to use the newly solved structure to, using computational approaches, analyze enzyme interactions with both bound substrate and bound product; the goal being to develop approaches for rationally designing improved cellulases for biomass deconstruction. We solved the structures of five cellulases from thermophilic fungi. Several of these were also solved with bound substrate/product, which allowed us to predict mutations that might enhance activity and stability.

  4. Degradation of lignocellulosic biomass and its subsequent utilization for the production of liquid fuels: Subcontract progress report, 1 March 1984-28 February 1985

    SciTech Connect (OSTI)

    Cooney, C.L.; Demain, A.L.; Sinskey, A.J.; Wang, D.I.C.

    1987-07-01

    This project is a coordinated effort to develop process technology for the degradation of lignocellulosic biomass and its utilization for the production of liquid fuels. Current efforts are based on our prior success in developing a single-step microbiological process for the conversion of lignocellulose to ethanol. This process utilizes a mixed culture of Clostridium thermocellum, a thermophilic celluloytic anaerobe which degrades cellulose and hemicellulose to fermentable sugars and Clostridium thermosaccharolyticum, a thermophilic anaerobe which produces high concentrations of ethanol from both hexoses and pentoses. These studies focus on the use of C. thermocellum and its cellulases for enhanced saccharification of lignocellulose and on the direct fermentation of lignocellulose to liquid fuel. Efforts on saccharification are directed to facilitate the adoption of existing fermentation ethanol plants for cellulosic substrates and to overcome the rate limiting step of saccharification in the mixed culture. 9 refs., 9 figs., 9 tabs.

  5. Mixed oxide nanoparticles and method of making

    DOE Patents [OSTI]

    Lauf, Robert J.; Phelps, Tommy J.; Zhang, Chuanlun; Roh, Yul

    2002-09-03

    Methods and apparatus for producing mixed oxide nanoparticulates are disclosed. Selected thermophilic bacteria cultured with suitable reducible metals in the presence of an electron donor may be cultured under conditions that reduce at least one metal to form a doped crystal or mixed oxide composition. The bacteria will form nanoparticles outside the cell, allowing easy recovery. Selection of metals depends on the redox potentials of the reducing agents added to the culture. Typically hydrogen or glucose are used as electron donors.

  6. Clostridium thermocellum Transcriptomic Profiles after Exposure to Furfural

    Office of Scientific and Technical Information (OSTI)

    or Heat Stress (Journal Article) | SciTech Connect Transcriptomic Profiles after Exposure to Furfural or Heat Stress Citation Details In-Document Search Title: Clostridium thermocellum Transcriptomic Profiles after Exposure to Furfural or Heat Stress Background The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP)biocatalyst for cellulosic ethanol production. It is capable of both cellulose solubilization and its fermentation to produce

  7. Innovations: Making Biofuels More Efficient | Department of Energy

    Broader source: Energy.gov (indexed) [DOE]

    Ginny Simmons Ginny Simmons Former Managing Editor for Energy.gov, Office of Public Affairs What are the key facts? Currently all biofuels rely on photosynthetic plants to convert energy from sunlight into usable fuel, but the overall efficiency of this is low. A new ARPA-E project is using thermophilic extremophiles -- microorganisms that grow optimally in temperatures above 160 deg F -- to produce a new highly efficient fuel. On Tuesday, Secretary Chu spoke of the need for new innovations to

  8. Microbial Reduction of Furfurals to Furan Alcohols by a Microbial Species -

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Energy Innovation Portal Microbial Reduction of Furfurals to Furan Alcohols by a Microbial Species Oak Ridge National Laboratory Contact ORNL About This Technology Technology Marketing SummaryAn ORNL researcher developed a method for producing furfuryl alcohol (FA) through bioprocessing using a thermophilic microorganism. This organism has been shown to be highly resistant to the toxic effects of furfural and hydroxymethylfurfural (HMF) and can propagate in the presence of over 48 g/L (500

  9. Nucleotide sequences encoding a thermostable alkaline protease

    DOE Patents [OSTI]

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  10. Nucleotide sequences encoding a thermostable alkaline protease

    DOE Patents [OSTI]

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  11. Deanne W. Sammond | Bioenergy | NREL

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Deanne W. Sammond Deanne W. Sammond Research Scientist Deanne.Sammond@nrel.gov | 303-384-6240 Research Interests Enhancing the conversion of biomass to fuel by improving the function of cellulases Consolidated bioprocessing-engineering thermophilic bacteria to produce ethanol Areas of Expertise Rational protein design Protein engineering Protein expression and purification Biophysical characterization of proteins Bioinformatics and structural analysis Education Ph.D., Department of Biochemistry

  12. Complete Genome Sequence of Clostridium clariflavum DSM 19732

    SciTech Connect (OSTI)

    Goodwin, Lynne A.; Davenport, Karen W.; Teshima, Hazuki; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Han, James; Pitluck, Sam; Nolan, Matt; Chen, Amy; Huntemann, Marcel; Mavromatis, K; Mikhailova, Natalia; Liolios, Konstantinos; Woyke, Tanja; Lynd, Lee R

    2012-01-01

    Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 proteincoding and 98 RNA genes, for the type strain DSM 19732.

  13. Sarah Hobdey 2012 Poster

    Office of Energy Efficiency and Renewable Energy (EERE) Indexed Site

    Saccharophagus degradans 2-40 (strain 2-40) is a cellulolytic marine bacterium that is has been shown to produce PHB from carbon rich environments, however, research has not ...

  14. Highest-Resolution Ribosome Structure

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    factors. Two structures of the intact ribosome from the common bacterium Escherichia coli, determined by a Berkeley-Berlin collaboration to a resolution of 3.5 , the highest...

  15. Methods for dispersing hydrocarbons using autoclaved bacteria

    DOE Patents [OSTI]

    Tyndall, R.L.

    1996-11-26

    A method of dispersing a hydrocarbon includes the following steps: providing a bacterium selected from the following group: ATCC 85527, ATCC 75529, and ATCC 55638, a mutant of any one of these bacteria possessing all the identifying characteristics of any one of these bacteria, and mixtures; autoclaving the bacterium to derive a dispersant solution; and contacting the dispersant solution with a hydrocarbon to disperse the hydrocarbon. Moreover, a method for preparing a dispersant solution includes the following steps: providing a bacterium selected from the following group: ATCC 75527, ATCC 75529, and ATCC 55638, a mutant of any one of these bacteria possessing all the identifying characteristics of any one of these bacteria, and mixtures; and autoclaving the bacterium to derive a dispersant solution.

  16. Methods for dispersing hydrocarbons using autoclaved bacteria

    DOE Patents [OSTI]

    Tyndall, Richard L.

    1996-01-01

    A method of dispersing a hydrocarbon includes the steps: providing a bacterium selected from the following group: ATCC 85527, ATCC 75529, and ATCC 55638, a mutant of any one of these bacteria possessing all the identifying characteristics of any one of these bacteria, and mixtures thereof; autoclaving the bacterium to derive a dispersant solution therefrom; and contacting the dispersant solution with a hydrocarbon to disperse the hydrocarbon. Moreover, a method for preparing a dispersant solution includes the following steps: providing a bacterium selected from the following group: ATCC 75527, ATCC 75529, and ATCC 55638, a mutant of any one of these bacteria possessing all the identifying characteristics of any one of these bacteria, and mixtures thereof; and autoclaving the bacterium to derive a dispersant solution therefrom.

  17. Crystal Structures of Anthrax Toxin Lethal Factor Bound to an...

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    bacterial capsule, is a major virulence factor in Anthrax1. The virulent strain of Bacillus anthracis is an encapsulated gram-positive, rod-shaped, spore-forming bacterium that...

  18. Structural Sequestration of Uranium in Bacteriogenic Manganese...

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    oxides precipitated around a spore (cell) of the marine Mn(II)-oxidizing bacterium, Bacillus sp., strain SG-1. This cell is about 0.5 m diameter (small axis). Manganese oxides...

  19. Structural Determination of Marine Bacteriogenic Manganese Oxides

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    oxides precipitated around a spore (cell) of the marine Mn(II)-oxidizing bac-terium, Bacillus sp., strain SG-1. This cell is about 0.5 m diameter (small axis). Bacterial...

  20. More on the term Fission? continued

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    particles.'" Rhodes quotes Arnold as saying, "Later that day Frisch looked me up and said, 'You work in a microbiology lab. What do you call the process in which one bacterium...

  1. LOS

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    N.M., April 19, 2013-New work from Los Alamos National Laboratory shows promise for stemming the advance of tuberculosis (TB) by revealing how the bacterium interacts with its...

  2. Advancing the art of tuberculosis detection

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    N.M., April 19, 2013-New work from Los Alamos National Laboratory shows promise for stemming the advance of tuberculosis (TB) by revealing how the bacterium interacts with its...

  3. Search for: All records | SciTech Connect

    Office of Scientific and Technical Information (OSTI)

    Have feedback or suggestions for a way to improve these results? Genome sequence of the ocean sediment bacterium Saccharomonospora marina type strain (XMU15T) Klenk, Hans-Peter ; ...

  4. Antimicrobial product and process

    DOE Patents [OSTI]

    Barrett, Karen B.

    1997-01-01

    A composition for controlling a plant disease caused by a plant pathogenic bacterium is disclosed. The composition comprises an activity for inhibiting the growth of the plant pathogenic bacterium and is extracted in an aqueous solvent from particles of malted cereal grain. The composition is used either in dry or wet form by application to plant parts, such as potato seed pieces, that are to be protected from the pathogenic bacteria.

  5. Antimicrobial product and process

    DOE Patents [OSTI]

    Barrett, K.B.

    1997-12-16

    A composition for controlling a plant disease caused by a plant pathogenic bacterium is disclosed. The composition comprises an activity for inhibiting the growth of the plant pathogenic bacterium and is extracted in an aqueous solvent from particles of malted cereal grain. The composition is used either in dry or wet form by application to plant parts, such as potato seed pieces, that are to be protected from the pathogenic bacteria. 6 figs.

  6. High ethanol producing derivatives of Thermoanaerobacter ethanolicus

    DOE Patents [OSTI]

    Ljungdahl, Lars G.; Carriera, Laura H.

    1983-01-01

    Derivatives of the newly discovered microorganism Thermoanaerobacter ethanolicus which under anaerobic and thermophilic conditions continuously ferment substrates such as starch, cellobiose, glucose, xylose and other sugars to produce recoverable amounts of ethanol solving the problem of fermentations yielding low concentrations of ethanol using the parent strain of the microorganism Thermoanaerobacter ethanolicus are disclosed. These new derivatives are ethanol tolerant up to 10% (v/v) ethanol during fermentation. The process includes the use of an aqueous fermentation medium, containing the substrate at a substrate concentration greater than 1% (w/v).

  7. High ethanol producing derivatives of Thermoanaerobacter ethanolicus

    DOE Patents [OSTI]

    Ljungdahl, L.G.; Carriera, L.H.

    1983-05-24

    Derivatives of the newly discovered microorganism Thermoanaerobacter ethanolicus which under anaerobic and thermophilic conditions continuously ferment substrates such as starch, cellobiose, glucose, xylose and other sugars to produce recoverable amounts of ethanol solving the problem of fermentations yielding low concentrations of ethanol using the parent strain of the microorganism Thermoanaerobacter ethanolicus are disclosed. These new derivatives are ethanol tolerant up to 10% (v/v) ethanol during fermentation. The process includes the use of an aqueous fermentation medium, containing the substrate at a substrate concentration greater than 1% (w/v).

  8. Combination biological and microwave treatments of used rubber products

    DOE Patents [OSTI]

    Fliermans, Carl B.; Wicks, George G.

    2002-01-01

    A process and resulting product is provided in which a vulcanized solid particulate, such as vulcanized crumb rubber, has select chemical bonds altered by biotreatment with thermophillic microorganisms selected from natural isolates from hot sulfur springs. Following the biotreatment, microwave radiation is used to further treat the surface and to treat the bulk interior of the crumb rubber. The resulting combined treatments render the treated crumb rubber more suitable for use in new rubber formulations. As a result, larger loading levels and sizes of the treated crumb rubber can be used in new rubber mixtures and good properties obtained from the new recycled products.

  9. Biological lignocellulose solubilization: Comparative evaluation of biocatalysts and enhancement via cotreatment

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Paye, Julie M. D.; Guseva, Anna; Hammer, Sarah K.; Gjersing, Erica; Davis, Mark F.; Davison, Brian H.; Olstad, Jessica; Donohoe, Bryon S.; Nguyen, Thanh Yen; Wyman, Charles E.; et al

    2016-01-12

    Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. Thus, to further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions.

  10. Production of extremophilic bacterial cellulase enzymes in aspergillus niger.

    SciTech Connect (OSTI)

    Gladden, John Michael

    2013-09-01

    Enzymes can be used to catalyze a myriad of chemical reactions and are a cornerstone in the biotechnology industry. Enzymes have a wide range of uses, ranging from medicine with the production of pharmaceuticals to energy were they are applied to biofuel production. However, it is difficult to produce large quantities of enzymes, especially if they are non-native to the production host. Fortunately, filamentous fungi, such as Aspergillus niger, are broadly used in industry and show great potential for use a heterologous enzyme production hosts. Here, we present work outlining an effort to engineer A. niger to produce thermophilic bacterial cellulases relevant to lignocellulosic biofuel production.

  11. Species profiles: Life histories and environmental requirements of coastal fishes and invertebrates (South Florida)

    SciTech Connect (OSTI)

    Zale, A.V.; Merrifield, S.G. )

    1989-07-01

    Species profiles are literature summaries of the taxonomy, morphology, distribution, life history, habitats, and environmental requirements of coastal species of fishes and aquatic invertebrates. They are designed to assist in environmental impact assessment. The tarpon and ladyfish are popular gamefishes. Adults spawn offshore. Larval and juvenile stages inhabit coastal marshes and mangroves. Both species are thermophilic (preferring warm water), euryhaline (tolerant of a wide range of salinity), and are capable of surviving at low oxygen concentrations. Wetlands destruction and degradation negatively affect these species by reducing nursery areas. 3 figs.

  12. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOE Patents [OSTI]

    Dees, H.C.

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  13. Cellulase producing microorganism ATCC 55702

    DOE Patents [OSTI]

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  14. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOE Patents [OSTI]

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  15. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOE Patents [OSTI]

    Dees, H. Craig

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  16. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOE Patents [OSTI]

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  17. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOE Patents [OSTI]

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  18. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOE Patents [OSTI]

    Dees, H.C.

    1998-07-14

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  19. Cellulase producing microorganism ATCC 55702

    DOE Patents [OSTI]

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  20. Biogenicity of silica precipitation around geysers and hot-spring vents, North Island, New Zealand

    SciTech Connect (OSTI)

    Jones, B.; Renaut, R.W.; Rosen, M.R.

    1997-01-01

    Before anthropogenic modifications, Ohaaki Pool (Broadlands-Ohaaki) and Dragon`s Mouth Geyser (Wairakei) emitted waters at temperatures of 93--98 C. The siliceous sinter that precipitated around their vents has the characteristics of geyserite, a dense laminated deposit of presumed abiogenic origin, that was precipitated from waters too hot (>73C) to support microbes other than thermophilic bacteria. Petrographic and SEM examinations of the sinters show that they incorporate columnar stromatolites and silicified, laminated stromatolitic mats that contain well-preserved filamentous microbes. At both localities the microbes lack evidence of desiccation or shrinkage, which implies that they were silicified rapidly at or shortly after their death. Although boiling and very hot (>90 C) waters were discharged, temperatures at many sites surrounding the vents remained sufficiently low and moist to support a microbial community that included thermophilic bacteria and cyanobacteria. In these cooler niches, the microbes and their biofilms served as highly favorable templates for the nucleation and growth of amorphous silica, and collectively provided a microbial framework for the laminated accretionary sinter. Some columnar, spicular, and stratiform geyserites are probably not abiotic precipitates, but are true silica stromatolites.

  1. Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments

    SciTech Connect (OSTI)

    Henriques, Barbara J. [Instituto Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras (Portugal); Saraiva, Ligia M. [Instituto Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras (Portugal); Gomes, Claudio M. [Instituto Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras (Portugal)]. E-mail: gomes@itqb.unl.pt

    2005-08-05

    Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T {sub opt} = 85 deg C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.

  2. Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities

    SciTech Connect (OSTI)

    Franke-Whittle, Ingrid H.; Walter, Andreas; Ebner, Christian; Insam, Heribert

    2014-11-15

    Highlights: • Different methanogenic communities in mesophilic and thermophilic reactors. • High VFA levels do not cause major changes in archaeal communities. • Real-time PCR indicated greater diversity than ANAEROCHIP microarray. - Abstract: A study was conducted to determine whether differences in the levels of volatile fatty acids (VFAs) in anaerobic digester plants could result in variations in the indigenous methanogenic communities. Two digesters (one operated under mesophilic conditions, the other under thermophilic conditions) were monitored, and sampled at points where VFA levels were high, as well as when VFA levels were low. Physical and chemical parameters were measured, and the methanogenic diversity was screened using the phylogenetic microarray ANAEROCHIP. In addition, real-time PCR was used to quantify the presence of the different methanogenic genera in the sludge samples. Array results indicated that the archaeal communities in the different reactors were stable, and that changes in the VFA levels of the anaerobic digesters did not greatly alter the dominating methanogenic organisms. In contrast, the two digesters were found to harbour different dominating methanogenic communities, which appeared to remain stable over time. Real-time PCR results were inline with those of microarray analysis indicating only minimal changes in methanogen numbers during periods of high VFAs, however, revealed a greater diversity in methanogens than found with the array.

  3. Anaerobic fermentation of woody biomass pretreated with supercritical ammonia

    SciTech Connect (OSTI)

    Weimer, P.J.; Chou, Y.C.T.

    1986-10-01

    The degradability of ground hardwood by thermophilic anaerobic bacteria (Clostridium thermocellum with or without Thermoanaerobacter strain B6A) was greatly enhanced by pretreatment of the substrate with supercritical ammonia. Relative to C. thermocellum monocultures, cocultures of C. thermocellum and Thermoanaerobacter strain B6A degraded 1.5-fold more pretreated soft maple but produced 2- 5-fold more fermentation end products because Thermoanaerobacter sp. removed reducing sugars produced by C. thermocellum during the fermentation. Dry weight losses were not totally accounted for in end products, due to formation of partially degraded material (<0.4 ..mu..m diameter wood particles) during the fermentation. One pretreated hardwood, Southern red oak, was fermented poorly because it released soluble inhibitors at the 60/sup 0/C incubation temperature. Considerable (6- to 11-fold) increases in substrate degradability were also noted for supercritical ammonia-pretreated wood materials fermented in an in vitro rumen digestibility assay. Degradation of pretreated softwoods by either thermophilic or mesophilic fermentation was not measurable under the conditions tested.

  4. Conversion of cellulose to ethanol by mesophilic bacteria. Progress report, July 15, 1983-February 15, 1985

    SciTech Connect (OSTI)

    Canale-Parola, E.

    1985-03-15

    Highlights of accomplishments during the period from July 1983 to February 1985 are summarized. Research has dealt primarily with strains of obligately anaerobic, mesophilic cellulolytic bacteria that we isolated from various natural environments. Eight strains (referred to as C strains) were isolated from mud of freshwater environments. As described in the previous progress report, the C strains represented a species of Clostridium that was different from other described species. The C strains fermented cellulose with formation of ethanol. They differed from thermophilic cellulolytic clostridia (e.g. Clostridium thermocellum) not only in growth temperature range, but also because they fermented xylan and pentoses with formation of ethanol. This result indicated that these mesophilic clostridia can convert to ethanol both cellulosic and hemicellulosic components of biomass. In contrast, monocultures of Clostridium thermocellum ferment only the cellulosic component of biomass. Furthermore, cellulose was degraded by the C strains at a rate comparable to that of thermophilic cellulolytic clostridia. These observations indicated that the mesophilic cellulolytic isolates constituted potentially useful microorganisms for ethanol production from biomass.

  5. Degradation of lignocellulosic biomass and its subsequent utilization for the production of liquid fuels: Subcontract progress report, 1 September 1981-28 February 1982

    SciTech Connect (OSTI)

    Cooney, C.L.; Demain, A.L.; Sinskey, A.J.; Wang, D.I.C.

    1987-07-01

    This project is a coordinated effort to develop process technology for the degradation of lignocellulosic biomass and its utilization for the production of liquid fuels. Current efforts are based on our prior success in developing a single-step microbiological process for the conversion of lignocellulose to ethanol. This process utilizes a mixed culture of Clostridium thermocellum, a thermophilic cellulolytic anaerobe which degrades cellulose and hemicellulose to fermentable sugars, and Clostridium thermosaccharolyticum, a thermophilic anaerobe which produces high concentrations of ethanol from both hexoses and pentoses. The proposed studies will focus on the use of C. thermocellum and its cellulases for enhanced saccharification of lignocellulose and on the direct fermentation of lignocellulose to the liquid fuel, butanol. Efforts on saccharification are directed to facilitate the adoption of existing fermentation ethanol plants for cellulosic substrates and to overcome the rate limiting step of saccharification in the mixed culture. The effort on butanol will extend the concept of direct fermentation to the production of this liquid fuel.

  6. Degradation of lignocellulosic biomass and its subsequent utilization for the production of liquid fuels: Subcontract progress report, 1 March 1981-31 August 1981

    SciTech Connect (OSTI)

    Cooney, C.L.; Demain, A.L.; Sinskey, A.J.; Wang, D.I.C.

    1987-07-01

    This project is a coordinated effort to develop process technology for the degradation of lignocellulosic biomass and its utilization for the production of liquid fuels. Current efforts are based on our prior success in developing a single-step microbiological process for the conversion of lignocellulose to ethanol. This process utilizes a mixed culture of Clostridium thermocellum, a thermophilic cellulolytic anaerobe which degrades cellulose and hemicellulose to fermentable sugars, and C. thermosaccharolyticum, a thermophilic anaerobe which produces high concentrations of ethanol from both hexoses and pentoses. The proposed studies will focus on the use of C. therocellum and its cellulases for enhanced saccharification of lignocellulose and on the direct fermentation of lignocellulose to the liquid fuel, butanol. Efforts on saccharification are directed to facilitate the adoption of existing fermentation ethanol plants for cellulosic substrates and to overcome the rate limiting step of saccharification in the mixed culture. The effort on butanol will extend the concept of direct fermentation to the production of this fuel. 55 figs., 6 tabs.

  7. Degradation of lignocellulosic biomass and its subsequent utilization for the production of liquid fuels: Subcontract progress report, 1 March 1982-31 August 1982

    SciTech Connect (OSTI)

    Cooney, C.L.; Demain, A.L.; Sinskey, A.J.; Wang, D.I.C.

    1987-07-01

    This project is a coordinated effort to develop process technology for the degradation of lignocellulosic biomass and its utilization for the production of liquid fuels. Current efforts are based on our prior success in developing a single-step microbiological process for the conversion of lignocellulose to ethanol. This process utilizes a mixed culture of Clostridium thermocellum, a thermophilic cellulolytic anaerobe which degrades cellulose and hemicellulose to fermentable sugars, and Clostridium thermosaccharolyticum, a thermophilic anaerobic which produces high concentrations of ethanol from both hexoses and pentoses. The proposed studies will focus on the use of C. thermocellum and its cellulases for enhanced saccharification of lignocellulose and on the direct fermentation of lignocellulose to the liquid fuel, butanol. Efforts on saccharification are directed to facilitate the adoption of existing fermentation ethanol plants for cellulosic substrates and to overcome the rate limiting step of saccharification in the mixed culture. The effort on butanol will extend the concept of direct fermentation to the production of this liquid fuel.

  8. Degradation of lignocellulosic biomass and its subsequent utilization for the production of liquid fuels: Subcontract progress report, 1 September 1982-28 February 1983

    SciTech Connect (OSTI)

    Cooney, C.L.; Demain, A.L.; Sinskey, A.J.; Wang, D.I.C.

    1987-07-01

    This project is a coordinated effort to develop process technology for the degradation of lignocellulosic biomass and its utilization for the production of liquid fuels. Current efforts are based on our prior success in developing a single-step microbiological process for the conversion of lignocellulose to ethanol. This process utilizes a mixed culture of Clostridium thermocellum, a thermophilic cellulolytic anaerobe which degrades cellulose and hemicellulose to fermentable sugars, and Clostridium thermosaccharolyticum, a thermophilic anaerobe which produces high concentrations of ethanol from both hexoses and pentoses. The proposed studies will focus on the use of C. thermocellum and its cellulases for enhanced saccharification of lignocellulose and on the direct fermentation of lignocellulose to the liquid fuel, butanol. Efforts on saccharification are directed to facilitate the adoption of existing fermentation ethanol plants for cellulosic substrates and to overcome the rate limiting step of saccharification in the mixed culture. The effort on butanol will extend the concept of direct fermentation to the production of this liquid fuel.

  9. Radiation-resistant microorganism

    DOE Patents [OSTI]

    Fliermans, Carl B.

    2010-06-15

    An isolated and purified bacterium is provided which was isolated from a high-level radioactive waste site of mixed waste. The isolate has the ability to degrade a wide variety of organic contaminants while demonstrating high tolerance to ionizing radiation. The organism is uniquely suited to bioremediation of a variety or organic contaminants while in the presence of ionizing radiation.

  10. Radiation-resistant microorganism

    DOE Patents [OSTI]

    Fliermans, Carl B.

    2007-01-09

    An isolated and purified bacterium is provided which was isolated from a high-level radioactive waste site of mixed waste. The isolate has the ability to degrade a wide variety of organic contaminants while demonstrating high tolerance to ionizing radiation. The organism is uniquely suited to bioremediation of a variety or organic contaminants while in the presence of ionizing radiation.

  11. Draft genome sequence of Streptomyces sp. strain Wb2n-11, a desert isolate with broad-spectrum antagonism against soilborne phytopathogens

    SciTech Connect (OSTI)

    Koeberl, Martina; White, Richard A.; Erschen, Sabine; El-Arabi, Tarek F.; Jansson, Janet K.; Berg, Gabriele

    2015-08-06

    Streptomyces sp. strain Wb2n-11, isolated from native desert soil, exhibited broad-spectrum antagonism against plant pathogenic fungi, bacteria and nematodes. The 8.2 Mb draft genome reveals genes putatively responsible for its promising biocontrol activity and genes which enable the soil bacterium to directly interact beneficially with plants.

  12. Production of amino acids using auxotrophic mutants of methylotrophic bacillus

    DOE Patents [OSTI]

    Hanson, Richard S.; Flickinger, Michael C.; Schendel, Frederick J.; Guettler, Michael V.

    2001-07-17

    A method of producing amino acids by culturing an amino acid auxotroph of a biologically pure strain of a type I methylotrophic bacterium of the genus Bacillus which exhibits sustained growth at 50.degree. C. using methanol as a carbon and energy source and requiring vitamin B.sub.12 and biotin is provided.

  13. Genome Sequence of a Chromium-Reducing Strain, Bacillus cereus S612

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Wang, Dongping; Boukhalfa, Hakim; Ware, Doug S.; Reimus, Paul W.; Daligault, Hajnalka E.; Gleasner, Cheryl D.; Johnson, Shannon L.; Li, Po-E

    2015-12-10

    We report here the genome sequence of an effective chromium-reducing bacterium,Bacillus cereusstrain S612. We found that the size of the draft genome sequence is approximately 5.4 Mb, with a G+C content of 35%, and it is predicted to contain 5,450 protein-coding genes.

  14. Draft Genome Sequence of Rhodococcus rhodochrous Strain ATCC 21198

    SciTech Connect (OSTI)

    Shields-Menard, Sara A.; Brown, Steven D; Klingeman, Dawn Marie; Indest, Karl; Hancock, Dawn; Wewalwela, Jayani; French, Todd; Donaldson, Janet

    2014-01-01

    Rhodococcus rhodochrous is a Gram-positive red-pigmented bacterium commonly found in the soil. The draft genome sequence for R. rhodochrous strain ATCC 21198 is presented here to provide genetic data for a better understanding of its lipid-accumulating capabilities.

  15. Genome Sequencing of 18 Francisella Strains To Aid in Assay Development and Testing

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; Coyne, Susan R.; Frey, Kenneth G.; Koroleva, Galina I.; Broomall, Stacey M.; Bishop-Lilly, Kimberly A.; Bruce, David C.; Chertkov, Olga; et al

    2015-04-30

    Francisella tularensis is a highly infectious bacterium that has the potential of causing high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

  16. Bioluminescent bioreporter integrated circuit devices and methods for detecting ammonia

    DOE Patents [OSTI]

    Simpson, Michael L [Knoxville, TN; Paulus, Michael J [Knoxville, TN; Sayler, Gary S [Blaine, TN; Applegate, Bruce M [West Lafayette, IN; Ripp, Steven A [Knoxville, TN

    2007-04-24

    Monolithic bioelectronic devices for the detection of ammonia includes a microorganism that metabolizes ammonia and which harbors a lux gene fused with a heterologous promoter gene stably incorporated into the chromosome of the microorganism and an Optical Application Specific Integrated Circuit (OASIC). The microorganism is generally a bacterium.

  17. Draft Genome Sequence of Streptomyces sp. Strain Wb2n-11, a Desert Isolate with Broad-Spectrum Antagonism against Soilborne Phytopathogens

    SciTech Connect (OSTI)

    Kberl, Martina; White, Richard A.; Erschen, Sabine; El-Arabi, Tarek F.; Jansson, Janet K.; Berg, Gabriele

    2015-08-06

    Streptomyces sp. strain Wb2n-11, isolated from native desert soil, exhibited broad-spectrum antagonism against plant pathogenic fungi, bacteria, and nematodes. The 8.2-Mb draft genome reveals genes putatively responsible for its promising biocontrol activity and genes which enable the soil bacterium to directly interact beneficially with plants.

  18. Bioremediation of nanomaterials

    DOE Patents [OSTI]

    Chen, Frank Fanqing; Keasling, Jay D; Tang, Yinjie J

    2013-05-14

    The present invention provides a method comprising the use of microorganisms for nanotoxicity study and bioremediation. In some embodiment, the microorganisms are bacterial organisms such as Gram negative bacteria, which are used as model organisms to study the nanotoxicity of the fullerene compounds: E. coli W3110, a human related enterobacterium and Shewanella oneidensis MR-1, an environmentally important bacterium with versatile metabolism.

  19. Complete Genome Sequence and Updated Annotation of Desulfovibrio alaskensis G20

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Hauser, Loren J.; Land, Miriam L.; Brown, Steven D.; Larimer, Frank L; Keller, Kimberly L.; Rapp-Giles, Barbara J.; Price, Morgan N.; Lin, Monica A.; Bruce, David C.; Detter, John C.; et al

    2011-06-17

    Desulfovibrio alaskensis G20 (formerly desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7 Mb genome sequence to provide insights into its physiology.

  20. Methods for targetted mutagenesis in gram-positive bacteria

    DOE Patents [OSTI]

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  1. Draft Genome Sequence of Streptomyces sp. Strain Wb2n-11, a Desert Isolate with Broad-Spectrum Antagonism against Soilborne Phytopathogens

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Köberl, Martina; White, Richard A.; Erschen, Sabine; El-Arabi, Tarek F.; Jansson, Janet K.; Berg, Gabriele

    2015-08-06

    Streptomyces sp. strain Wb2n-11, isolated from native desert soil, exhibited broad-spectrum antagonism against plant pathogenic fungi, bacteria, and nematodes. The 8.2-Mb draft genome reveals genes putatively responsible for its promising biocontrol activity and genes which enable the soil bacterium to directly interact beneficially with plants.

  2. Neutron Reflectometry and QCM-D Study of the Interaction of Cellulase Enzymes with Films of Amorphous Cellulose

    SciTech Connect (OSTI)

    Halbert, Candice E; Ankner, John Francis; Kent, Michael S; Jaclyn, Murton K; Browning, Jim; Cheng, Gang; Liu, Zelin; Majewski, Jaroslaw; Supratim, Datta; Michael, Jablin; Bulent, Akgun; Alan, Esker; Simmons, Blake

    2011-01-01

    Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation (QCM-D) of the interaction of a commercial fungal enzyme extract (T. viride), two purified endoglucanses from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima), and a mesophilic fungal endoglucanase (Cel45A from H. insolens) with amorphous cellulose films. The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. NR reveals the profile of water through the film at nm resolution, while QCM-D provides changes in mass and film stiffness. At 20 oC and 0.3 mg/ml, the T. viride cocktail rapidly digested the entire film, beginning from the surface followed by activity throughout the bulk of the film. For similar conditions, Cel9A and Cel5A were active for only a short period of time and only at the surface of the film, with Cel9A releasing 40 from the ~ 700 film and Cel5A resulting in only a slight roughening/swelling effect at the surface. Subsequent elevation of the temperature to the Topt in each case resulted in a very limited increase in activity, corresponding to the loss of an additional 60 from the film for Cel9A and 20 from the film for Cel5A, and very weak penetration into and digestion within the bulk of the film, before the activity again ceased. The results for Cel9A and Cel5A contrast sharply with results for Cel45A where very rapid and extensive penetration and digestion within the bulk of the film was observed at 20 C. We speculate that the large differences are due to the use of the thermophilic enzymes far below their optimal temperatures and also the presence of a cellulose binding module (CBM) on Cel45A while the thermophilic enzymes lack a CBM.

  3. Complete genome sequence of Hippea maritima type strain (MH2T)

    SciTech Connect (OSTI)

    Huntemann, Marcel; Lu, Megan; Nolan, Matt; Lapidus, Alla L.; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxanne; Han, Cliff; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Pagani, Ioanna; Ivanova, N; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Detter, J. Chris; Brambilla, Evelyne-Marie; Rohde, Manfred; Spring, Stefan; Goker, Markus; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Mavromatis, K

    2011-01-01

    Hippea maritima (Miroshnichenko et al. 1999) is the type species of the genus Hippea, which belongs to the family Desulfurellaceae within the class Deltaproteobacteria. The anaerobic, moderately thermophilic marine sulfur-reducer was first isolated from shallow-water hot vents in Matipur Harbor, Papua New Guinea. H. maritima was of interest for genome se- quencing because of its isolated phylogenetic location, as a distant next neighbor of the ge- nus Desulfurella. Strain MH2T is the first type strain from the order Desulfurellales with a com- pletely sequenced genome. The 1,694,430 bp long linear genome with its 1,723 protein- coding and 57 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  4. In situ thermally enhanced biodegradation of petroleum fuel hydrocarbons and halogenated organic solvents

    DOE Patents [OSTI]

    Taylor, Robert T.; Jackson, Kenneth J.; Duba, Alfred G.; Chen, Ching-I

    1998-01-01

    An in situ thermally enhanced microbial remediation strategy and a method for the biodegradation of toxic petroleum fuel hydrocarbon and halogenated organic solvent contaminants. The method utilizes nonpathogenic, thermophilic bacteria for the thermal biodegradation of toxic and carcinogenic contaminants, such as benzene, toluene, ethylbenzene and xylenes, from fuel leaks and the chlorinated ethenes, such as trichloroethylene, chlorinated ethanes, such as 1,1,1-trichloroethane, and chlorinated methanes, such as chloroform, from past solvent cleaning practices. The method relies on and takes advantage of the pre-existing heated conditions and the array of delivery/recovery wells that are created and in place following primary subsurface contaminant volatilization efforts via thermal approaches, such as dynamic underground steam-electrical heating.

  5. In situ thermally enhanced biodegradation of petroleum fuel hydrocarbons and halogenated organic solvents

    DOE Patents [OSTI]

    Taylor, R.T.; Jackson, K.J.; Duba, A.G.; Chen, C.I.

    1998-05-19

    An in situ thermally enhanced microbial remediation strategy and a method for the biodegradation of toxic petroleum fuel hydrocarbon and halogenated organic solvent contaminants are described. The method utilizes nonpathogenic, thermophilic bacteria for the thermal biodegradation of toxic and carcinogenic contaminants, such as benzene, toluene, ethylbenzene and xylenes, from fuel leaks and the chlorinated ethenes, such as trichloroethylene, chlorinated ethanes, such as 1,1,1-trichloroethane, and chlorinated methanes, such as chloroform, from past solvent cleaning practices. The method relies on and takes advantage of the pre-existing heated conditions and the array of delivery/recovery wells that are created and in place following primary subsurface contaminant volatilization efforts via thermal approaches, such as dynamic underground steam-electrical heating. 21 figs.

  6. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    SciTech Connect (OSTI)

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  7. Closing the Carbon Balance for Fermentation by Clostridium thermocellum (ATCC 27405)

    SciTech Connect (OSTI)

    Ellis, Lucas D; Holwerda, Evert K; Hogsett, David; Rogers, Steve; Shao, Xiongjun; Tschaplinski, Timothy J; Thorne, Phil; Lynd, L.

    2012-01-01

    Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, {>=}92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products - ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.

  8. Saccharification of complex cellulosic substrates by the cellulase system from Clostridium thermocellum

    SciTech Connect (OSTI)

    Johnson, E.A.; Sakajoh, M.; Halliwell, G.; Madia, A.; Demain, A.L.

    1982-05-01

    True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca/sup 2 +/ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70/sup 0/C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70/sup 0/C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.

  9. Solvent Immersion Imprint Lithography

    SciTech Connect (OSTI)

    Vasdekis, Andreas E.; Wilkins, Michael J.; Grate, Jay W.; Kelly, Ryan T.; Konopka, Allan; Xantheas, Sotiris S.; Chang, M. T.

    2014-06-21

    The mechanism of polymer disolution was explored for polymer microsystem prototyping, including microfluidics and optofluidics. Polymer films are immersed in a solvent, imprinted and finally brought into contact with a non-modified surface to permanently bond. The underlying polymer-solvent interactions were experimentally and theoretically investigated, and enabled rapid polymer microsystem prototyping. During imprinting, small molecule integration in the molded surfaces was feasible, a principle applied to oxygen sensing. Polystyrene (PS) was employed for microbiological studies at extreme environmental conditions. The thermophile anaerobe Clostridium Thermocellum was grown in PS pore-scale micromodels, revealing a double mean generation lifetime than under ideal culture conditions. Microsystem prototyping through directed polymer dissolution is simple and accessible, while simultaneous patterning, bonding, and surface/volume functionalization are possible in less than one minute.

  10. Microbiology and physiology of anaerobic fermentations of cellulose

    SciTech Connect (OSTI)

    Wiegel, J.

    1991-05-01

    The biochemistry and physiology of four major groups of anaerobic bacteria involved in the conversion of cellulose to methane or chemical feedstocks are examined. Aspects of metabolism which are relevant to the interactions and bioenergetics of consortia are being studied. Properties of the cellulolytic enzyme cluster of Clostridium thermocellum are investigated. Five different hydrogenases have been characterized in detail from anaerobic bacteria. Genes for different hydrogenases are being cloned and sequenced to determine their structural relationships. The role of metal clusters in activation of H{sub 2} is being investigated, as is the structure and role of metal clusters in formate metabolism. The function of formate in the total synthesis of acetate from CO{sub 2} and the role of this primary in anaerobes will be examined as well. Finally, these enzyme studies will be performed on thermophilic bacteria and new, pertinent species will be isolated. 50 refs., 3 figs., 1 tab.

  11. Saccharification of complex cellulosic substrates by the cellulase system from Clostridium thermocellum

    SciTech Connect (OSTI)

    Johnson, E.A.; Sakajoh, M.; Halliwell, G.; Madia, A.; Demain, A.L.

    1982-05-01

    True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of CA/sub 2//sup +/ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70 degrees C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70 degrees C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity. (Refs. 26).

  12. Co-digestion of cattle manure with food waste and sludge to increase biogas production

    SciTech Connect (OSTI)

    Maranon, E.; Castrillon, L.; Quiroga, G.; Fernandez-Nava, Y.; Gomez, L.; Garcia, M.M.

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer Small increase in methane production was observed applying sonication pretreatment. Black-Right-Pointing-Pointer Biogas productions between 720 and 1100 mL/Lreactor day were achieved. Black-Right-Pointing-Pointer Volatile solids removal efficiencies ranged between 53% and 60%. Black-Right-Pointing-Pointer Lower methane yields were obtained when operating under thermophilic conditions. Black-Right-Pointing-Pointer Optimum OLR in lab-scale CSTR was 1.2-1.3 g VS/L day (HRT: 20 days). - Abstract: Anaerobic co-digestion strategies are needed to enhance biogas production, especially when treating certain residues such as cattle/pig manure. This paper presents a study of co-digestion of cattle manure with food waste and sewage sludge. With the aim of maximising biogas yields, a series of experiments were carried out under mesophilic and thermophilic conditions using continuously stirred-tank reactors, operating at different hydraulic residence times. Pretreatment with ultrasound was also applied to compare the results with those obtained with non-pretreated waste. Specific methane production decreases when increasing the OLR and decreasing HRT. The maximum value obtained was 603 LCH{sub 4}/kg VS{sub feed} for the co-digestion of a mixture of 70% manure, 20% food waste and 10% sewage sludge (total solid concentration around 4%) at 36 Degree-Sign C, for an OLR of 1.2 g VS/L day. Increasing the OLR to 1.5 g VS/L day led to a decrease of around 20-28% in SMP. Lower methane yields were obtained when operating at 55 Degree-Sign C. The increase in methane production when applying ultrasound to the feed mixtures does not compensate for the energy spent in this pretreatment.

  13. Zymomonas mobilis - Science and industrial application

    SciTech Connect (OSTI)

    Doelle, H.W.; Kirk, L.; Crittenden, R.; Toh, Hsien ); Doelle, M.B. )

    1993-01-01

    Zymomonas mobilis is undoubtedly one of the most unique bacterium within the microbial world. Known since 1912 under the names Termobacterium mobilis, Pseudomonas linderi, and Zymomonas mobilis, reviews on its uniqueness have been published in 1977 and 1988. The bacterium zymomonas mobilis not only exhibits an extraordinarily uniqueness in its biochemistry, but also in its growth behavior, energy production, and response to culture conditions, as well as cultivation techniques used. This uniqueness caused great interest in the scientific, biotechnological, and industrial worlds. Its ability to couple and uncouple energy production in favor of product formation, to respond to physical and chemical environment manipulation, as well as its restricted product formation, makes it an ideal microorganism for microbial process development. This review explores the advances made since 1987, together with new developments in the pure scientific and applied commercial areas. 362 refs.

  14. In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

    SciTech Connect (OSTI)

    Spano, Anthony J. . E-mail: ajs6z@virginia.edu; Chen, Frank S.; Goodman, Benjamin E.; Sabat, Agnes E.; Simon, Martha N.; Wall, Joseph S.; Correia, John J.; McIvor, Wilson; Newcomb, William W.; Brown, Jay C.; Schnur, Joel M.; Lebedev, Nikolai

    2007-07-20

    The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass {approx} 4.3 MDa, representing 101 {+-} 11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.

  15. 1

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Mysteries of 'molecular machines' revealed December 22, 2014 EMBARGOED for Monday, December 22, 11 a.m. Eastern Time Phenix software uses X-ray diffraction spots to produce 3-D image LOS ALAMOS, N.M., Dec. 22, 2014-Scientists are making it easier for pharmaceutical companies and researchers to see the detailed inner workings of molecular machines. "Inside each cell in our bodies and inside every bacterium and virus are tiny but complex protein molecules that synthesize chemicals, replicate

  16. Highest-Resolution Ribosome Structure

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Highest-Resolution Ribosome Structure Print The last step in converting the genetic information stored in DNA into the major functional parts of cells is protein biosynthesis. Protein synthesis occurs on the ribosome, a cellular factory found in all forms of life. In contrast to most cellular machines, the ribosome contains a functional core of RNA that is enhanced by ribosomal proteins and accessory factors. Two structures of the intact ribosome from the common bacterium Escherichia coli,

  17. Investigating the role of CheA-3 in Dusulfovibrio Vulgaris Hildenborough

    Office of Scientific and Technical Information (OSTI)

    (Technical Report) | SciTech Connect Investigating the role of CheA-3 in Dusulfovibrio Vulgaris Hildenborough Citation Details In-Document Search Title: Investigating the role of CheA-3 in Dusulfovibrio Vulgaris Hildenborough Multiple sets of chemotaxis genes including three cheA homologs were identified in the genome sequence of the anaerobic bacterium Desulfovibrio vulgaris Hildenborough. Each CheA is a histidine kinase (HK) and part of a two component signal transduction system. Knock out

  18. Genome Sequence of a Chromium-Reducing Strain, Bacillus cereus S612

    SciTech Connect (OSTI)

    Wang, Dongping; Boukhalfa, Hakim; Ware, Doug S.; Reimus, Paul W.; Daligault, Hajnalka E.; Gleasner, Cheryl D.; Johnson, Shannon L.; Li, Po-E

    2015-12-10

    We report here the genome sequence of an effective chromium-reducing bacterium,Bacillus cereusstrain S612. We found that the size of the draft genome sequence is approximately 5.4 Mb, with a G+C content of 35%, and it is predicted to contain 5,450 protein-coding genes.

  19. LOS

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Advancing the art of tuberculosis detection April 19, 2013 LOS ALAMOS, N.M., April 19, 2013-New work from Los Alamos National Laboratory shows promise for stemming the advance of tuberculosis (TB) by revealing how the bacterium interacts with its human hosts and thus providing a new pathway for early detection in patients.A recent publication from the Los Alamos Biosensor Team describes the association of a key tuberculosis virulence factor, lipoarabinomannan (LAM) with human high-density

  20. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Substrate Recognition Strategy for Botulinum Neurotoxin Print Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. By inhibiting release of the neurotransmitter acetylcholine, for example, the neurotoxin produced by the bacterium Clostridium botulinum interferes with nerve impulses and causes a paralysis of respiratory and skeletal muscles that can cause death. Researchers from Stanford University have now determined the first structure of a

  1. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Substrate Recognition Strategy for Botulinum Neurotoxin Print Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. By inhibiting release of the neurotransmitter acetylcholine, for example, the neurotoxin produced by the bacterium Clostridium botulinum interferes with nerve impulses and causes a paralysis of respiratory and skeletal muscles that can cause death. Researchers from Stanford University have now determined the first structure of a

  2. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Substrate Recognition Strategy for Botulinum Neurotoxin Print Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. By inhibiting release of the neurotransmitter acetylcholine, for example, the neurotoxin produced by the bacterium Clostridium botulinum interferes with nerve impulses and causes a paralysis of respiratory and skeletal muscles that can cause death. Researchers from Stanford University have now determined the first structure of a

  3. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Substrate Recognition Strategy for Botulinum Neurotoxin Substrate Recognition Strategy for Botulinum Neurotoxin Print Wednesday, 25 May 2005 00:00 Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. By inhibiting release of the neurotransmitter acetylcholine, for example, the neurotoxin produced by the bacterium Clostridium botulinum interferes with nerve impulses and causes a paralysis of respiratory and skeletal muscles that can cause

  4. A Key Enzyme to the Potency of an Anticancer Agent

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    A Key Enzyme to the Potency of an Anticancer Agent Print Incorporation of halogen atoms into drug molecules often increases biological activity. This is the case with salinosporamide A (sal A), a natural product from the marine bacterium Salinispora tropica that is 500 times more active than sal B, its nonchlorinated analog. Sal A is in phase I human clinical trials for the treatment of multiple myeloma and solid tumors. A group of researchers, using diffraction data collected at ALS Beamline

  5. A Key Enzyme to the Potency of an Anticancer Agent

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    A Key Enzyme to the Potency of an Anticancer Agent Print Incorporation of halogen atoms into drug molecules often increases biological activity. This is the case with salinosporamide A (sal A), a natural product from the marine bacterium Salinispora tropica that is 500 times more active than sal B, its nonchlorinated analog. Sal A is in phase I human clinical trials for the treatment of multiple myeloma and solid tumors. A group of researchers, using diffraction data collected at ALS Beamline

  6. A Key Enzyme to the Potency of an Anticancer Agent

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    A Key Enzyme to the Potency of an Anticancer Agent Print Incorporation of halogen atoms into drug molecules often increases biological activity. This is the case with salinosporamide A (sal A), a natural product from the marine bacterium Salinispora tropica that is 500 times more active than sal B, its nonchlorinated analog. Sal A is in phase I human clinical trials for the treatment of multiple myeloma and solid tumors. A group of researchers, using diffraction data collected at ALS Beamline

  7. Draft Genome Sequence of thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 Reveals Molecular Adaptations to Extreme pH and Temperature

    SciTech Connect (OSTI)

    Kalamorz, Falk; Keis, Stefanie; Stanton, Jo-Ann; Brown, Steven D; Klingeman, Dawn Marie; Land, Miriam L; Han, Cliff; Martin, S L.; Morgan, Hugh; Cook, Greg

    2011-01-01

    The genes and molecular machines that allow for a thermoalkaliphilic lifestyle have not been defined. To address this goal, we report on the improved high-quality draft genome sequence of Caldalkalibacillus thermarum strain TA2.A1, an obligately aerobic bacterium that grows optimally at pH 9.5 and 65 to 70 C on a wide variety of carbon and energy sources.

  8. Stories of Discovery & Innovation: A Step Toward Artificial Photosynthesis

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    | U.S. DOE Office of Science (SC) A Step Toward Artificial Photosynthesis Energy Frontier Research Centers (EFRCs) EFRCs Home Centers Research Science Highlights News & Events EFRC News EFRC Events DOE Announcements Publications History Contact BES Home 01.06.12 Stories of Discovery & Innovation: A Step Toward Artificial Photosynthesis Print Text Size: A A A Subscribe FeedbackShare Page EFRC researchers construct an artificial version of a bacterium's light-absorbing

  9. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Structural Basis for Activation of Cholera Toxin Print Cholera is a serious disease that claims thousands of victims each year in third-world, war-torn, and disaster-stricken nations. The culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it secretes cholera toxin (CT), a protein whose A1 subunit (CTA1) triggers a series of events culminating in the massive efflux of

  10. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Structural Basis for Activation of Cholera Toxin Print Cholera is a serious disease that claims thousands of victims each year in third-world, war-torn, and disaster-stricken nations. The culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it secretes cholera toxin (CT), a protein whose A1 subunit (CTA1) triggers a series of events culminating in the massive efflux of

  11. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Structural Basis for Activation of Cholera Toxin Print Cholera is a serious disease that claims thousands of victims each year in third-world, war-torn, and disaster-stricken nations. The culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it secretes cholera toxin (CT), a protein whose A1 subunit (CTA1) triggers a series of events culminating in the massive efflux of

  12. Larry Taylor | Bioenergy | NREL

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Larry Taylor Research Scientist Larry.Taylor@nrel.gov | 303-384-7784 Research Interests Larry Taylor received his Ph.D. in Environmental Molecular Biology and Biotechnology from the Marine and Estuarine Environmental Sciences department at the University of Maryland, College Park. His dissertation work focused on the functional genomics of the plant cell wall degrading enzyme systems of the marine bacterium Saccharophagus degradans 2-40, which was isolated from decaying salt marsh grass in the

  13. News Item

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Self-photosensitization of Nonphotosynthetic Bacteria for Solar-to-Chemical Production M. thermoacetica-CdS hybrids are formed by the one-pot growth and biological precipitation of CdS nanoparticles which serve as light absorbers for photosynthesis. Scientific Achievement Molecular Foundry users induced the nonphotosynthetic, CO2 reducing bacterium M. thermoacetica to precipitate cadmium sulfide nanoparticles which serve as light harvesters to enable photosynthetic production of acetic acid.

  14. Purple Bacteria Develops Its Own Form of Sunscreen | U.S. DOE Office of

    Office of Science (SC) Website

    Science (SC) Purple Bacteria Develops Its Own Form of "Sunscreen" Energy Frontier Research Centers (EFRCs) EFRCs Home Centers Research Science Highlights Highlight Archives News & Events Publications History Contact BES Home 05.03.12 Purple Bacteria Develops Its Own Form of "Sunscreen" Print Text Size: A A A FeedbackShare Page Scientific Achievement Found that specific pigments in the light harvesting complex of a photosynthetic bacterium act primarily to protect the

  15. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Structural Basis for Activation of Cholera Toxin Print Cholera is a serious disease that claims thousands of victims each year in third-world, war-torn, and disaster-stricken nations. The culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it secretes cholera toxin (CT), a protein whose A1 subunit (CTA1) triggers a series of events culminating in the massive efflux of

  16. Substrate Recognition Strategy for Botulinum Neurotoxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Substrate Recognition Strategy for Botulinum Neurotoxin Print Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. By inhibiting release of the neurotransmitter acetylcholine, for example, the neurotoxin produced by the bacterium Clostridium botulinum interferes with nerve impulses and causes a paralysis of respiratory and skeletal muscles that can cause death. Researchers from Stanford University have now determined the first structure of a

  17. Systems biology analysis of Zymomonas mobilis ZM4 ethanol stress responses

    Office of Scientific and Technical Information (OSTI)

    (Journal Article) | SciTech Connect Systems biology analysis of Zymomonas mobilis ZM4 ethanol stress responses Citation Details In-Document Search Title: Systems biology analysis of Zymomonas mobilis ZM4 ethanol stress responses Zymomonas mobilis ZM4 is a capable ethanogenic bacterium with high ethanol productivity and high level of ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol

  18. Anthrax Lethal Factor

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Thiang Yian Wong, Robert Schwarzenbacher and Robert C. Liddington The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Anthrax Toxin is a major virulence factor in the infectious disease, Anthrax1. This toxin is produced by Bacillus anthracis, which is an encapsulated, spore-forming, rod-shaped bacterium. Inhalation anthrax, the most deadly form, is contracted through breathing spores. Once spores germinate within cells of the immune system called macrophages2, bacterial

  19. Draft genome sequence of Therminicola potens strain JR

    SciTech Connect (OSTI)

    Byrne-Bailey, K.G.; Wrighton, K.C.; Melnyk, R.A.; Agbo, P.; Hazen, T.C.; Coates, J.D.

    2010-07-01

    'Thermincola potens' strain JR is one of the first Gram-positive dissimilatory metal-reducing bacteria (DMRB) for which there is a complete genome sequence. Consistent with the physiology of this organism, preliminary annotation revealed an abundance of multiheme c-type cytochromes that are putatively associated with the periplasm and cell surface in a Gram-positive bacterium. Here we report the complete genome sequence of strain JR.

  20. A biotemplated nickel nanostructure: Synthesis, characterization and antibacterial activity

    SciTech Connect (OSTI)

    Ashtari, Khadijeh; Fasihi, Javad; Mollania, Nasrin; Khajeh, Khosro

    2014-02-01

    Highlights: Nickel nanostructure-encapsulated bacteria were prepared using electroless deposition. Bacterium surface was activated by red-ox reaction of its surface amino acids. Interfacial changes at cell surfaces were investigated using fluorescence spectroscopy. TEM and AFM depicted morphological changes. Antibacterial activity of nanostructure was examined against different bacteria strains. - Abstract: Nickel nanostructure-encapsulated bacteria were prepared using the electroless deposition procedure and activation of bacterium cell surface by red-ox reaction of surface amino acids. The electroless deposition step occurred in the presence of Ni(II) and dimethyl amine boran (DMAB). Interfacial changes at bacteria cell surfaces during the coating process were investigated using fluorescence spectroscopy. Fluorescence of tryptophan residues was completely quenched after the deposition of nickel onto bacteria surfaces. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) depicted morphological changes on the surface of the bacterium. It was found that the Ni coated nanostructure was mechanically stable after ultrasonication for 20 min. Significant increase in surface roughness of bacteria was also observed after deposition of Ni clusters. The amount of coated Ni on the bacteria surface was calculated as 36% w/w. The antibacterial activity of fabricated nanostructure in culture media was examined against three different bacteria strains; Escherichia coli, Bacillus subtilis and Xantomonas campestris. The minimum inhibitory concentrations (MIC) were determined as 500 mg/L, 350 mg/L and 200 mg/L against bacteria, respectively.

  1. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; Engle, Nancy L.; Tschaplinski, Timothy J.; Hettich, Robert L.; Elkins, James G.

    2014-12-03

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr- and isobutyraldehydes. Conclusions: Thermoanaerobacter pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomics analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.« less

  2. Efficient breakdown of lignocellulose using mixed-microbe populations for bioethanol production.

    SciTech Connect (OSTI)

    Murton, Jaclyn K.; Ricken, James Bryce; Powell, Amy Jo

    2009-11-01

    This report documents progress in discovering new catalytic technologies that will support the development of advanced biofuels. The global shift from petroleum-based fuels to advanced biofuels will require transformational breakthroughs in biomass deconstruction technologies, because current methods are neither cost effective nor sufficiently efficient or robust for scaleable production. Discovery and characterization of lignocellulolytic enzyme systems adapted to extreme environments will accelerate progress. Obvious extreme environments to mine for novel lignocellulolytic deconstruction technologies include aridland ecosystems (ALEs), such as those of the Sevilleta Long Term Ecological Research (LTER) site in central New Mexico (NM). ALEs represent at least 40% of the terrestrial biosphere and are classic extreme environments, with low nutrient availability, high ultraviolet radiation flux, limited and erratic precipitation, and extreme variation in temperatures. ALEs are functionally distinct from temperate environments in many respects; one salient distinction is that ALEs do not accumulate soil organic carbon (SOC), in marked contrast to temperate settings, which typically have large pools of SOC. Low productivity ALEs do not accumulate carbon (C) primarily because of extraordinarily efficient extracellular enzyme activities (EEAs) that are derived from underlying communities of diverse, largely uncharacterized microbes. Such efficient enzyme activities presumably reflect adaptation to this low productivity ecosystem, with the result that all available organic nutrients are assimilated rapidly. These communities are dominated by ascomycetous fungi, both in terms of abundance and contribution to ecosystem-scale metabolic processes, such as nitrogen and C cycling. To deliver novel, robust, efficient lignocellulolytic enzyme systems that will drive transformational advances in biomass deconstruction, we have: (1) secured an award through the Department of Energy (DoE) Joint Genome Institute (JGI) to perform metatranscriptomic functional profiling of eukaryotic microbial communities of blue grama grass (Bouteloua gracilis) rhizosphere (RHZ) soils and (2) isolated and provided initial genotypic and phenotypic characterization data for thermophilic fungi. Our preliminary results show that many strains in our collection of thermophilic fungi frequently outperform industry standards in key assays; we also demonstrated that this collection is taxonomically diverse and phenotypically compelling. The studies summarized here are being performed in collaboration with University of New Mexico and are based at the Sevilleta LTER research site.

  3. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    SciTech Connect (OSTI)

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; Engle, Nancy L.; Tschaplinski, Timothy J.; Hettich, Robert L.; Elkins, James G.

    2014-12-03

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr- and isobutyraldehydes. Conclusions: Thermoanaerobacter pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomics analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

  4. 1

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Rapid diagnosis a new weapon against re-emerging TB June 7, 2015 Science on the Hill: Rapid diagnosis a new weapon against re-emerging TB Around the world, tuberculosis is making a comeback, owing to the increased incidence of HIV/AIDS and several other factors. The untreatable drug-resistant strains of the bacterium are rapidly increasing, causing grave concern. Drug resistance is a widespread global challenge today and could result in a post-antibiotic era, if unchecked. That and the global

  5. 1

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Counting small RNA in disease-causing organisms June 17, 2013 Small molecules of RNA (tens to hundreds of nucleotides in length) play a key regulatory role in bacteria. Due to their small size, directly measuring the number of small RNA (sRNA) present in a single bacterium has proven so far to be an impossible task. Standard methods of measuring the number of specific nucleic acid molecules present in a single cell suffer from too much background and false positives when scientists attempt to

  6. Klebsiella pneumoniae inoculants for enhancing plant growth

    DOE Patents [OSTI]

    Triplett, Eric W.; Kaeppler, Shawn M.; Chelius, Marisa K.

    2008-07-01

    A biological inoculant for enhancing the growth of plants is disclosed. The inoculant includes the bacterial strains Herbaspirillum seropedicae 2A, Pantoea agglomerans P101, Pantoea agglomerans P102, Klebsiella pneumoniae 342, Klebsiella pneumoniae zmvsy, Herbaspirillum seropedicae Z152, Gluconacetobacter diazotrophicus PA15, with or without a carrier. The inoculant also includes strains of the bacterium Pantoea agglomerans and K. pneumoniae which are able to enhance the growth of cereal grasses. Also disclosed are the novel bacterial strains Herbaspirillum seropedicae 2A, Pantoea agglomerans P101 and P102, and Klebsiella pneumoniae 342 and zmvsy.

  7. Anaerobic dehalogenation of hydroxylated polychlorinated biphenyls by Desulfitobacterium dehalogenans

    SciTech Connect (OSTI)

    Wiegel, J.; Zhang, X.; Wu, Q.

    1999-05-01

    Ten years after reports on the existence of anaerobic dehalogenation of polychlorinated biphenyls (PCBs) in sediment slurries, the authors report here on the rapid reductive dehalogenation of para-hydroxylated PCBs (HO-PCBs), the excreted main metabolites of PCB in mammals, which can exhibit estrogenic and antiestrogenic activities in humans. The anaerobic bacterium Desulfitobacterium dehalogenans completely dehalogenates all flanking chlorines (chlorines in ortho position to the para-hydroxyl group) from congeners such as 3,3{prime},5,5{prime}-tetrachloro-4,4{prime}-dihydroxybiphenyl.

  8. October 2, 2008: NETL and Zebra mussels | Department of Energy

    Office of Energy Efficiency and Renewable Energy (EERE) Indexed Site

    , 2008: NETL and Zebra mussels October 2, 2008: NETL and Zebra mussels October 2, 2008: NETL and Zebra mussels October 2, 2008 The Department announces that DOE-funded researchers have developed an environmentally safe bacterial toxin to control zebra and quagga mussels, two non-native, invasive species that have found their way into the waterways of 25 states over the past two decades, fouling the aquatic environment as they spread. The new bio-pesticide was derived from a common soil bacterium

  9. A Key Enzyme to the Potency of an Anticancer Agent

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    A Key Enzyme to the Potency of an Anticancer Agent A Key Enzyme to the Potency of an Anticancer Agent Print Wednesday, 28 May 2008 00:00 Incorporation of halogen atoms into drug molecules often increases biological activity. This is the case with salinosporamide A (sal A), a natural product from the marine bacterium Salinispora tropica that is 500 times more active than sal B, its nonchlorinated analog. Sal A is in phase I human clinical trials for the treatment of multiple myeloma and solid

  10. Structural Basis for Activation of Cholera Toxin

    Broader source: All U.S. Department of Energy (DOE) Office Webpages (Extended Search)

    Structural Basis for Activation of Cholera Toxin Structural Basis for Activation of Cholera Toxin Print Wednesday, 30 November 2005 00:00 Cholera is a serious disease that claims thousands of victims each year in third-world, war-torn, and disaster-stricken nations. The culprit is the bacterium Vibrio cholerae, which can be ingested through contaminated food or water and colonizes the mucous membrane of the human small intestine. There, it secretes cholera toxin (CT), a protein whose A1 subunit

  11. Final report for DOE grant FG02-06ER15805

    SciTech Connect (OSTI)

    Daniel Gage

    2012-05-31

    DOE funding was used to investigate the role of the phosphotransferase system (PTS) in the symbiotic, nodulating bacterium Sinorhizobium meliloti. This system is well studied in several bacterial species. However, it??s organization and function in S. meliloti is substantially different than in the those other, well-studied bacteria. The S. meliloti PTS, through our DOE-funded work, has become a model for how this important signal transduction system works in the a-proteobacteria. We have found that the PTS is relatively simple, used for only signal transduction and not transport, and is involved in regulation of carbon metabolism in response to carbon availability and nitrogen availability.

  12. Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Sander, Kyle B.; Wilson, Charlotte M.; M. Rodriquez, Jr.; Klingeman, Dawn Marie; Davison, Brian H.; Brown, Steven D.; Rydzak, T.

    2015-12-12

    Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. As a result, towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.

  13. Annotation of the Clostridium Acetobutylicum Genome

    SciTech Connect (OSTI)

    Daly, M. J.

    2004-06-09

    The genome sequence of the solvent producing bacterium Clostridium acetobutylicum ATCC824, has been determined by the shotgun approach. The genome consists of a 3.94 Mb chromosome and a 192 kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases, closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria.

  14. MOONSHINER I: personal fuel production. Final report

    SciTech Connect (OSTI)

    Holloman, R.L.

    1981-01-01

    This report describes the research and design of a self-controlling cellulose to liquid fuel conversion reactor. Initial research suggested the possibility of utilization of a bacterium named Clostridium thermocellum as a conversion agent due to its unique metabolism. Further research showed that work at other locations supported that possibility. Work was begun on the apparatus and techniques necessary for completion. Bad technique, design or supplies resulted in many months of ineffectual work while progress was being made on similar research elsewhere. Other projects' data was used to continue the information collection and design stages of this effort.

  15. Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation

    SciTech Connect (OSTI)

    Sander, Kyle B.; Wilson, Charlotte M.; M. Rodriquez, Jr.; Klingeman, Dawn Marie; Davison, Brian H.; Brown, Steven D.; Rydzak, T.

    2015-12-12

    Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. As a result, towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.

  16. Environmentally Safe Control of Zebra Mussel Fouling

    SciTech Connect (OSTI)

    Daniel Molloy

    2008-02-29

    The two primary objectives of this USDOE-NETL contract were successfully achieved during the project: (1) to accelerate research on the development of the bacterium Pseudomonas fluorescens strain CL145A (Pf-CL145A) as a biocontrol agent for zebra mussels (Dreissena polymorpha) and quagga mussels (Dreissena rostriformis bugensis)--two invasive freshwater bivalve species that are infesting water pipes in power plants; and (2) to identify a private-sector company that would move forward to commercialize Pf-CL145A as a substitute for the current polluting use of biocide chemicals for control of these dreissenid mussels in power plant pipes.

  17. Biogasification of sorghum in a novel anaerobic digester

    SciTech Connect (OSTI)

    Srivastava, V.J.; Biljetina, R.; Isaacson, H.R.; Hayes, T.D.

    1987-01-01

    The Institute of Gas Technology (IGT) conducted pilot-scale anaerobic digestion experiments with ensiled sorghum in a 160 ft/sup 3/ digester at the experimental test unit (ETU) facility at the Walt Disney World Resort Complex in Florida. The study focused on improving bioconversion efficiencies and process stability by employing a novel reactor concept developed at IGT. Steady-state performance data were collected from the ETU as well as from a laboratory-scale conventional stirred tank reactor (CSTR) at loading rates of 0.25 and 0.50 lb organic matter/ft/sup 3/-day at mesophilic and thermophilic temperatures, respectively. This paper will describe the ETU facility, novel digester design and operating techniques, and the results obtained during 12 months of stable and uninterrupted operation of the ETU and the CSTR which showed that methane yields anad rates from the ETU were 20% to 50% higher than those of the CSTR. 10 refs., 7 figs., 5 tabs.

  18. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

    SciTech Connect (OSTI)

    Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D'haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

    2009-11-15

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  19. Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

    SciTech Connect (OSTI)

    Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

    2011-05-11

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  20. Structures of the Signal Recognition Particle Receptor From the Archaeon Pyrococcus Furiosus: Implications for the Targeting Step at the Membrane

    SciTech Connect (OSTI)

    Egea, P.F.; Tsuruta, H.; Leon, G.P.de; Napetschnig, J.; Walter, P.; Stroud, R.M.

    2009-05-18

    In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP {center_dot} magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP {center_dot} SR targeting complexes.

  1. Nutrient requirements and growth physiology of the photoheterotrophic Acidobacterium, Chloracidobacterium thermophilum

    SciTech Connect (OSTI)

    Tank, Marcus; Bryant, Donald A.

    2015-03-27

    A novel thermophilic, microaerophilic, anoxygenic, and chlorophototrophic member of the phylum Acidobacteria, Chloracidobacterium thermophilum strain BT, was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, Wyoming. C. thermophilum is strictly dependent on light and oxygen and grows optimally as a photoheterotroph at irradiance values between 20 and 50 mol photons m? s?. C. thermophilum is unable to synthesize branched-chain amino acids (AAs), L-lysine, and vitamin B??, which are required for growth. Although the organism lacks genes for autotrophic carbon fixation, bicarbonate is also required. Mixtures of other AAs and 2-oxoglutarate stimulate growth. As suggested from genomic sequence data, C. thermophilum requires a reduced sulfur source such as thioglycolate, cysteine, methionine, or thiosulfate. The organism can be grown in a defined medium at 51 C (Topt; range 4458C) in the pH range 5.59.5 (pHopt = ~7.0). Using the defined growth medium and optimal conditions, it was possible to isolate new C. thermophilum strains directly from samples of hot springs mats in Yellowstone National Park, Wyoming. The new isolates differ from the type strain with respect to pigment composition, morphology in liquid culture, and temperature adaptation.

  2. Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1

    SciTech Connect (OSTI)

    Akioka, Makoto [Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522 (Japan); Nakano, Hiroaki [Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo, Kyoto 606-8501 (Japan); Horikiri, Aya; Tsujimoto, Yoshiyuki; Matsui, Hiroshi [Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522 (Japan); Shimizu, Tetsuya; Nakatsu, Toru; Kato, Hiroaki [Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo, Kyoto 606-8501 (Japan); Watanabe, Kunihiko, E-mail: kwatanab@kpu.ac.jp [Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522 (Japan)

    2006-12-01

    Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out. To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 . A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 202.1 from the MAD data set from the SeMet-substituted crystal were used for phase determination.

  3. Multiple Syntrophic Interactions in a Terephthalate-Degrading Methanogenic Consortium

    SciTech Connect (OSTI)

    Lykidis, Athanasios; Chen, Chia-Lung; Tringe, Susannah G.; McHardy, Alice C.; Copeland, Alex 5; Kyrpides, Nikos C.; Hugenholtz, Philip; Liu, Wen-Tso

    2010-08-05

    Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium tinside a hyper-mesophilic (i.e., between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified ?-oxidation to H{sub 2}/CO{sub 2} and acetate. These intermediates are converted to CH{sub 4}/CO{sub 2} by three novel hyper-mesophilic methanogens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to COsub 2}/H{sub 2} and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H{sub 2}-producing syntroph ? methanogen partnership that may serve to improve community stability.

  4. Factors controlling pathogen destruction during anaerobic digestion of biowastes

    SciTech Connect (OSTI)

    Smith, S.R. . E-mail: s.r.smith@imperial.ac.uk; Lang, N.L.; Cheung, K.H.M.; Spanoudaki, K.

    2005-07-01

    Anaerobic digestion is the principal method of stabilising biosolids from urban wastewater treatment in the UK, and it also has application for the treatment of other types of biowaste. Increasing awareness of the potential risks to human and animal health from environmental sources of pathogens has focused attention on the efficacy of waste treatment processes at destroying pathogenic microorganisms in biowastes recycled to agricultural land. The degree of disinfection achieved by a particular anaerobic digester is influenced by a variety of interacting operational variables and conditions, which can often deviate from the ideal. Experimental investigations demonstrate that Escherichia coli and Salmonella spp. are not damaged by mesophilic temperatures, whereas rapid inactivation occurs by thermophilic digestion. A hydraulic, biokinetic and thermodynamic model of pathogen inactivation during anaerobic digestion showed that a 2 log{sub 10} reduction in E. coli (the minimum removal required for agricultural use of conventionally treated biosolids) is likely to challenge most conventional mesophilic digesters, unless strict maintenance and management practices are adopted to minimise dead zones and by-pass flow. Efficient mixing and organic matter stabilisation are the main factors controlling the rate of inactivation under mesophilic conditions and not a direct effect of temperature per se on pathogenic organisms.

  5. Nutrient requirements and growth physiology of the photoheterotrophic Acidobacterium, Chloracidobacterium thermophilum

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Tank, Marcus; Bryant, Donald A.

    2015-03-27

    A novel thermophilic, microaerophilic, anoxygenic, and chlorophototrophic member of the phylum Acidobacteria, Chloracidobacterium thermophilum strain BT, was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, Wyoming. C. thermophilum is strictly dependent on light and oxygen and grows optimally as a photoheterotroph at irradiance values between 20 and 50 µmol photons m⁻² s⁻¹. C. thermophilum is unable to synthesize branched-chain amino acids (AAs), L-lysine, and vitamin B₁₂, which are required for growth. Although the organism lacks genes for autotrophic carbon fixation, bicarbonate is also required. Mixtures of other AAs and 2-oxoglutarate stimulatemore » growth. As suggested from genomic sequence data, C. thermophilum requires a reduced sulfur source such as thioglycolate, cysteine, methionine, or thiosulfate. The organism can be grown in a defined medium at 51° C (Topt; range 44–58°C) in the pH range 5.5–9.5 (pHopt = ~7.0). Using the defined growth medium and optimal conditions, it was possible to isolate new C. thermophilum strains directly from samples of hot springs mats in Yellowstone National Park, Wyoming. The new isolates differ from the type strain with respect to pigment composition, morphology in liquid culture, and temperature adaptation.« less

  6. Characterization of Clostridium thermocellum strains with disrupted fermentation end product pathways

    SciTech Connect (OSTI)

    Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D; Johnson, Courtney M; Tschaplinski, Timothy J; Martin, Madhavi Z; Engle, Nancy L; Argyros, Aaron; Van den Berg, Robert A; Caiazza, Nicky; Guss, Adam M; Lynd, Lee R

    2013-01-01

    Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of Llactate ( ldh) and/or acetate ( pta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end products. Cellobiose-grown cultures of the ldh strain had identical biomass accumulation, fermentation end products, transcription profile and intracellular metabolite concentrations compared to its parent strain (DSM1313 hpt spo0A). The pta-deficient strain grew slower and had 30% lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A ldh pta double mutant strain evolved for faster growth had growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to pta. Free amino acids were secreted by all examined strains, with both pta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for ldh pta reached 5 mM by the end of growth, or 2.7% of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to 6-fold in the pta and 16-fold in the ldh pta strain. We hypothesize that the deletions in fermentation end product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP+ through increased production of amino acids.

  7. Degradation of lignocellulosic biomass and its subsequent utilization for the production of liquid fuels: Subcontract progress report, 1 March 1983-29 February 1984

    SciTech Connect (OSTI)

    Cooney, C.L.; Demain, A.L.; Sinskey, A.J.; Wang, D.I.C.

    1987-07-01

    This project is a coordinated effort to develop process technology for the degradation of lignocellulosic biomass and its utilization for the production of liquid fuels. Current efforts are based on our prior success in developing a single-step microbiological process for the conversion of lignocellulose to ethanol. This process utilizes a mixed culture of Clostridium thermocellum, a thermophilic cellulolytic anaerobe which degrades cellulose and hemicellulose to fermentable sugars, and Clostridium thermosaccharolyticum, a thermo anaerobe which produces high concentrations of ethanol from both hexoses and pentoses. The proposed studies will focus on the use of C. thermocellum and its cellulases for enhanced saccharification of lignocellulose and on the direct fermentation of lignocellulose to the liquid fuel, butanol. Efforts on saccharification are directed to facilitate the adoption of existing fermentation ethanol plants for cellulosic substrates and to overcome the rate limiting step of saccharification in the mixed culture. The effort on butanol will extend the concept of direct fermentation to the production of this liquid fuel. 14 refs.

  8. Purification and crystallization of a trimodular complex comprising the type II cohesin–dockerin interaction from the cellulosome of Clostridium thermocellum

    SciTech Connect (OSTI)

    Adams, Jarrett J.; Pal, Gour; Yam, Katherine; Spencer, Holly L.; Jia, Zongchao; Smith, Steven P.

    2005-01-01

    A trimodular complex comprising the type II cohesin–dockerin interaction from the cellulosome of C. thermocellum has been purified and crystallized by the hanging-drop vapour-diffusion method. A native crystal and a selenomethionine derivative have been analyzed using X-ray diffraction. The high-affinity calcium-mediated type II cohesin–dockerin interaction is responsible for the attachment of the multi-enzyme cellulose-degrading complex, termed the cellulosome, to the cell surface of the thermophilic anaerobe Clostridium thermocellum. A trimodular 40 kDa complex comprising the SdbA type II cohesin and the the CipA type II dockerin–X module modular pair from the cellulosome of C. thermocellum has been crystallized. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 45.21, b = 52.34, c = 154.69 Å. The asymmetric unit contains one molecule of the protein complex and native and selenomethionine-derivative crystals diffracted to 2.1 and 2.0 Å, respectively.

  9. Increase in ethanol yield via elimination of lactate production in an ethanol-tolerant mutant of Clostridium thermocellum

    SciTech Connect (OSTI)

    Biswas, Ranjita; Prabhu, Sandeep; Lynd, Lee R; Guss, Adam M

    2014-01-01

    Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) ldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) ldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.

  10. Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

    SciTech Connect (OSTI)

    Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D; Johnson, Courtney M; Tschaplinski, Timothy J; Martin, Madhavi Z; Engle, Nancy L; Van den Berg, Robert A; Argyros, Aaron; Caiazza, Nicky; Guss, Adam M; Lynd, Lee R

    2013-01-01

    Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Dldh) and/or acetate (Dpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Dldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Dhpt Dspo0A). The Dpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A Dldh Dpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Dpta. Free amino acids were secreted by all examined strains, with both Dpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Dldh Dpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Dpta and 16-fold in the Dldh Dpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP* through increased production of amino acids.

  11. Biogasification of sorghum

    SciTech Connect (OSTI)

    Biljetina, R.; Srivastava, V.J.; Isaacson, H.R.

    1987-01-01

    The Institute of Gas Technology has been operating a 1200-gallon, anaerobic solids-concentrating digester at the Walt Disney World Resort Complex in Lake Buena Vista, Florida. This digester development work is part of a larger effort sponsored by the Gas Research Institute to provide an effective community waste treatment and energy recovery concept for smaller communities. As a result, an economically attractive, water hyacinth-based wastewater treatment system was developed that includes the digestion of water hyacinth and sludge to methane. A further extension of the community waste treatment concept is to include agricultural wastes in the energy recovery scheme. Therefore, during 1986 a test program was initiated to obtain data on the digestion of sorghum in the solids concentrating digester. Performance data was collected at both mesophilic and thermophilic operating conditions at total organic loading rates of 0.25 and 0.5 pounds per cubic foot of digester volume per day, respectively. Excellent methane yields were obtained during twelve months of stable and uninterrupted operation. This paper summarizes the performance data obtained on sorghum in this digester. 7 refs., 6 figs., 6 tabs.

  12. Establishment of stable synthetic mutualism without co-evolution between microalgae and bacteria demonstrated by mutual transfer of metabolites (NanoSIMS isotopic imaging) and persistent physical association (Fluorescent in situ hybridization)

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    de-Bashan, Luz E.; Mayali, Xavier; Bebout, Brad M.; Weber, Peter K.; Detweiler, Angela M.; Hernandez, Juan- Pablo; Prufert-Bebout, Leslie; Bashan, Yoav

    2016-03-03

    The demonstration of a mutualistic interaction requires evidence of benefits for both partners as well as stability of the association over multiple generations. A synthetic mutualism between the freshwater microalga Chlorella sorokiniana and the soil-derived plant growth-promoting bacterium (PGPB) Azospirillum brasilense was created when both microorganisms were co-immobilized in alginate beads. Using stable isotope enrichment experiments followed by high-resolution secondary ion mass spectrometry (SIMS) imaging of single cells, we demonstrated transfer of carbon and nitrogen compounds between the two partners. Further, using fluorescent in situ hybridization (FISH), mechanical disruption and scanning electron microscopy, we demonstrated the stability of their physicalmore » association for a period of 10 days after the aggregated cells were released from the beads. The bacteria significantly enhanced the growth of the microalgae while the microalgae supported growth of the bacteria in a medium where it could not otherwise grow. In conclusion, we propose that this microalga-bacterium association is a true synthetic mutualism independent of co-evolution. (155 words).« less

  13. Investigation of the chemical interface in the soybean–aphid and rice–bacteria interactions using MALDI-mass spectrometry imaging

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Klein, Adam T.; Yagnik, Gargey B.; Hohenstein, Jessica D.; Ji, Zhiyuan; Zi, Jiachen; Reichert, Malinda D.; MacIntosh, Gustavo C.; Yang, Bing; Peters, Reuben J.; Vela, Javier; et al

    2015-04-27

    Mass spectrometry imaging (MSI) is an emerging technology for high-resolution plant biology. It has been utilized to study plant–pest interactions, but limited to the surface interfaces. Here we expand the technology to explore the chemical interactions occurring inside the plant tissues. Two sample preparation methods, imprinting and fracturing, were developed and applied, for the first time, to visualize internal metabolites of leaves in matrix-assisted laser desorption ionization (MALDI)-MSI. This is also the first time nanoparticle-based ionization was implemented to ionize diterpenoid phytochemicals that were difficult to analyze with traditional organic matrices. The interactions between rice–bacterium and soybean–aphid were investigated asmore » two model systems to demonstrate the capability of high-resolution MSI based on MALDI. Localized molecular information on various plant- or pest-derived chemicals provided valuable insight for the molecular processes occurring during the plant–pest interactions. Basically, salicylic acid and isoflavone based resistance was visualized in the soybean–aphid system and antibiotic diterpenoids in rice–bacterium interactions.« less

  14. Investigation of the chemical interface in the soybeanaphid and ricebacteria interactions using MALDI-mass spectrometry imaging

    SciTech Connect (OSTI)

    Klein, Adam T.; Yagnik, Gargey B.; Hohenstein, Jessica D.; Ji, Zhiyuan; Zi, Jiachen; Reichert, Malinda D.; MacIntosh, Gustavo C.; Yang, Bing; Peters, Reuben J.; Vela, Javier; Lee, Young Jin

    2015-04-27

    Mass spectrometry imaging (MSI) is an emerging technology for high-resolution plant biology. It has been utilized to study plantpest interactions, but limited to the surface interfaces. Here we expand the technology to explore the chemical interactions occurring inside the plant tissues. Two sample preparation methods, imprinting and fracturing, were developed and applied, for the first time, to visualize internal metabolites of leaves in matrix-assisted laser desorption ionization (MALDI)-MSI. This is also the first time nanoparticle-based ionization was implemented to ionize diterpenoid phytochemicals that were difficult to analyze with traditional organic matrices. The interactions between ricebacterium and soybeanaphid were investigated as two model systems to demonstrate the capability of high-resolution MSI based on MALDI. Localized molecular information on various plant- or pest-derived chemicals provided valuable insight for the molecular processes occurring during the plantpest interactions. Basically, salicylic acid and isoflavone based resistance was visualized in the soybeanaphid system and antibiotic diterpenoids in ricebacterium interactions.

  15. Stable zymomonas mobilis xylose and arabinose fermenting strains

    DOE Patents [OSTI]

    Zhang, Min; Chou, Yat-Chen

    2008-04-08

    The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

  16. Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

    SciTech Connect (OSTI)

    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

  17. Nonphotochemical Hole-Burning Studies of Energy Transfer Dynamics in Antenna Complexes of Photosynthetic Bacteria

    SciTech Connect (OSTI)

    Satoshi Matsuzaki

    2002-08-01

    This thesis contains the candidate's original work on excitonic structure and energy transfer dynamics of two bacterial antenna complexes as studied using spectral hole-burning spectroscopy. The general introduction is divided into two chapters (1 and 2). Chapter 1 provides background material on photosynthesis and bacterial antenna complexes with emphasis on the two bacterial antenna systems related to the thesis research. Chapter 2 reviews the underlying principles and mechanism of persistent nonphotochemical hole-burning (NPHB) spectroscopy. Relevant energy transfer theories are also discussed. Chapters 3 and 4 are papers by the candidate that have been published. Chapter 3 describes the application of NPHB spectroscopy to the Fenna-Matthews-Olson (FMO) complex from the green sulfur bacterium Prosthecochloris aestuarii; emphasis is on determination of the low energy vibrational structure that is important for understanding the energy transfer process associated within three lowest energy Qy-states of the complex. The results are compared with those obtained earlier on the FMO complex from Chlorobium tepidum. In Chapter 4, the energy transfer dynamics of the B800 molecules of intact LH2 and B800-deficient LH2 complexes of the purple bacterium Rhodopseudomonas acidophila are compared. New insights on the additional decay channel of the B800 ring of bacteriochlorophyll a (BChl a) molecules are provided. General conclusions are given in Chapter 5.

  18. MICROBIAL FERMENTATION OF ABUNDANT BIOPOLYMERS: CELLULOSE AND CHITIN

    SciTech Connect (OSTI)

    Leschine, Susan

    2009-10-31

    Our research has dealt with seven major areas of investigation: i) characterization of cellulolytic members of microbial consortia, with special attention recently given to Clostridium phytofermentans, a bacterium that decomposes cellulose and produces uncommonly large amounts of ethanol, ii) investigations of the chitinase system of Cellulomonas uda; including the purification and characterization of ChiA, the major component of this enzyme system, iii) molecular cloning, sequence and structural analysis of the gene that encodes ChiA in C. uda, iv) biofilm formation by C. uda on nutritive surfaces, v) investigations of the effects of humic substances on cellulose degradation by anaerobic cellulolytic microbes, vi) studies of nitrogen metabolism in cellulolytic anaerobes, and vii) understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. Also, progress toward completing the research of more recent projects is briefly summarized. Major accomplishments include: 1. Characterization of Clostridium phytofermentans, a cellulose-fermenting, ethanol-producing bacterium from forest soil. The characterization of a new cellulolytic species isolated from a cellulose-decomposing microbial consortium from forest soil was completed. This bacterium is remarkable for the high concentrations of ethanol produced during cellulose fermentation, typically more than twice the concentration produced by other species of cellulolytic clostridia. 2. Examination of the use of chitin as a source of carbon and nitrogen by cellulolytic microbes. We discovered that many cellulolytic anaerobes and facultative aerobes are able to use chitin as a source of both carbon and nitrogen. This major discovery expands our understanding of the biology of cellulose-fermenting bacteria and may lead to new applications for these microbes. 3. Comparative studies of the cellulase and chitinase systems of Cellulomonas uda. Results of these studies indicate that the chitinase and cellulase systems of this bacterium are distinct in terms of the proteins involved and the regulation of their production. 4. Characterization of the chitinase system of C. uda. A 70,000-Mr endochitinase, designated ChiA, was purified from C. uda culture supernatant fluids and characterized. 5. Analysis of chiA, which codes for the major enzymatic component of the chitinase system of C. uda. The gene encoding the endochitinase ChiA in C. uda was cloned, its complete nucleotide sequence was determined and its implications were investigated. 6. Formation of biofilms by C. uda on cellulose and chitin. Microscopic observations indicated that, under conditions of nitrogen limitation, C. uda cells grew as a biofilm attached tightly to the surface of cellulose or chitin. 7. Development of tools for a genetic approach to studies of cellulose fermentation by cellulolytic clostridia. We have explored the potential of various techniques, and obtained evidence indicating that Tn916 mutagenesis may be particularly effective in this regard. As part of this research, we identified the presence of a plasmid in one strain, which was cloned, sequenced, and analyzed for its utility in the development of vectors for genetic studies. 8. Effects of humic substances on cellulose degradation by anaerobic cellulolytic microbes. We determined that humic substances play an important role in the anaerobic cellulose decomposition and in the physiology of cellulose-fermenting soil bacteria. 9. Nitrogenases of cellulolytic clostridia. We described a nitrogenase gene from a cellulolytic clostridium and presented evidence, based on sequence analyses and conserved gene order, for lateral gene transfer between this bacterium and a methanogenic archaeon. 10. Characterization of Clostridium hungatei, a new N2-fixing cellulolytic species isolated from a methanogenic consortium from soil. 11. Understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. We discovered that C. papyrosolvens produces a multiprotein, multicomplex cellulase-xylanase enzyme system that hydrolyzes crystalline cellulose, and we have described this system in detail.

  19. Final Report - "CO2 Sequestration in Cell Biomass of Chlorobium Thiosulfatophilum"

    SciTech Connect (OSTI)

    James L. Gaddy, PhD; Ching-Whan Ko, PhD

    2009-05-04

    World carbon dioxide emissions from the combustion of fossil fuels have increased at a rate of about 3 percent per year during the last 40 years to over 24 billion tons today. While a number of methods have been proposed and are under study for dealing with the carbon dioxide problem, all have advantages as well as disadvantages which limit their application. The anaerobic bacterium Chlorobium thiosulfatophilum uses hydrogen sulfide and carbon dioxide to produce elemental sulfur and cell biomass. The overall objective of this project is to develop a commercial process for the biological sequestration of carbon dioxide and simultaneous conversion of hydrogen sulfide to elemental sulfur. The Phase I study successfully demonstrated the technical feasibility of utilizing this bacterium for carbon dioxide sequestration and hydrogen sulfide conversion to elemental sulfur by utilizing the bacterium in continuous reactor studies. Phase II studies involved an advanced research and development to develop the engineering and scale-up parameters for commercialization of the technology. Tasks include culture isolation and optimization studies, further continuous reactor studies, light delivery systems, high pressure studies, process scale-up, a market analysis and economic projections. A number of anaerobic and aerobic microorgansims, both non-photosynthetic and photosynthetic, were examined to find those with the fastest rates for detailed study to continuous culture experiments. C. thiosulfatophilum was selected for study to anaerobically produce sulfur and Thiomicrospira crunogena waws selected for study to produce sulfate non-photosynthetically. Optimal conditions for growth, H2S and CO2 comparison, supplying light and separating sulfur were defined. The design and economic projections show that light supply for photosynthetic reactions is far too expensive, even when solar systems are considered. However, the aerobic non-photosynthetic reaction to produce sulfate with T. crunogena produces a reasonable return when treating a sour gas stream of 120 million SCFD containing 2.5 percent H2S. In this case, the primary source of revenue is from desulfurization of the gas stream. While the technology has significant application in sequestering carbon dioxide in cell biomass or single cell proten (SCP), perhaps the most immediate application is in desulfurizing LGNG or other gas streams. This biological approach is a viable economical alternative to existing hydrogen sulfide removal technology, and is not sensitive to the presence of hydrocarbons which act as catalyst poisons.

  20. An active sitetail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

    SciTech Connect (OSTI)

    Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

    2015-09-23

    Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 resolution contains tailactive site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that close the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an open structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active sitetail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

  1. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

  2. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    SciTech Connect (OSTI)

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

  3. The Genome of Syntrophomonas Wolfei: New Insights into Syntrophic Metabolism and Biohydrogen Production

    SciTech Connect (OSTI)

    Sieber, Jessica R.; Sims, David R.; Han, Cliff F.; Kim, E.; Lykidis, Athanasios; Lapidus, Alla; McDonald, Erin; Rohlin, Lars; Culley, David E.; Gunsalus, Robert; McInerney, Michael J.

    2010-08-01

    Syntrophomonas wolfei is a specialist, evolutionarily adapted for syntrophic growth with methanogens and other hydrogen- and/or formate-using microorganisms. This slow growing anaerobe has three putative ribosome RNA operons, each of which has 16S rRNA and 23S rRNA genes of different length and multiple 5S rRNA genes. The genome also contains ten RNA-directed, DNA polymerase genes. Genomic analysis shows that S. wolfei relies solely on the reduction of protons, bicarbonate, or unsaturated fatty acids to re-oxidize reduced cofactors. S. wolfei lacks the genes needed for aerobic or anaerobic respiration and has an exceptionally limited ability to create ion gradients. An ATP synthase and a pyrophosphatase were the only systems detected capable of creating an ion gradient. Multiple homologs for ?-oxidation genes were present even though S. wolfei uses a limited range of fatty acids from 4 to 8 carbons in length. S. wolfei, other syntrophic metabolizers with completed genomic sequences, and thermophilic anaerobes known to produce high molar ratios of hydrogen from glucose have genes to produce H2 from NADH by an electron bifurcation mechanism. Comparative genomic analysis also suggests that formate production from NADH may involve electron bifurcation. A membrane-bound, iron-sulfur oxidoreductase found in S. wolfei and Syntrophus aciditrophicus may be uniquely involved in reverse electron transport during syntrophic fatty acid metabolism. The genome sequence of S. wolfei reveals several core reactions that may be characteristic of syntrophic fatty acid metabolism and illustrates how biological systems produce hydrogen from thermodynamically difficult reactions.

  4. Heterologous production of an energy-conserving carbon monoxide dehydrogenase complex in the hyperthermophile Pyrococcus furiosus

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-29

    In this study, carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificialmore » chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100° C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80° C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.« less

  5. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

    SciTech Connect (OSTI)

    Onstott, T. C.; Aubrey, A.D.; Kieft, T L; Silver, B J; Phelps, Tommy Joe; Van Heerden, E.; Opperman, D. J.; Bada, J L.

    2014-01-01

    Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 C and 1 2 years for 3 km depth and 54 C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

  6. Archaeal community composition affects the function of anaerobic co-digesters in response to organic overload

    SciTech Connect (OSTI)

    Lerm, S.; Kleyboecker, A.; Miethling-Graff, R.; Alawi, M.; Kasina, M.; Liebrich, M.; Wuerdemann, H.

    2012-03-15

    Highlights: Black-Right-Pointing-Pointer Two types of methanogens are necessary to respond successfully to perturbation. Black-Right-Pointing-Pointer Diversity of methanogens correlates with the VFA concentration and methane yield. Black-Right-Pointing-Pointer Aggregates indicate tight spatial relationship between minerals and microorganisms. - Abstract: Microbial community diversity in two thermophilic laboratory-scale and three full-scale anaerobic co-digesters was analysed by genetic profiling based on PCR-amplified partial 16S rRNA genes. In parallel operated laboratory reactors a stepwise increase of the organic loading rate (OLR) resulted in a decrease of methane production and an accumulation of volatile fatty acids (VFAs). However, almost threefold different OLRs were necessary to inhibit the gas production in the reactors. During stable reactor performance, no significant differences in the bacterial community structures were detected, except for in the archaeal communities. Sequencing of archaeal PCR products revealed a dominance of the acetoclastic methanogen Methanosarcina thermophila, while hydrogenotrophic methanogens were of minor importance and differed additionally in their abundance between reactors. As a consequence of the perturbation, changes in bacterial and archaeal populations were observed. After organic overload, hydrogenotrophic methanogens (Methanospirillum hungatei and Methanoculleus receptaculi) became more dominant, especially in the reactor attributed by a higher OLR capacity. In addition, aggregates composed of mineral and organic layers formed during organic overload and indicated tight spatial relationships between minerals and microbial processes that may support de-acidification processes in over-acidified sludge. Comparative analyses of mesophilic stationary phase full-scale reactors additionally indicated a correlation between the diversity of methanogens and the VFA concentration combined with the methane yield. This study demonstrates that the coexistence of two types of methanogens, i.e. hydrogenotrophic and acetoclastic methanogens is necessary to respond successfully to perturbation and leads to stable process performance.

  7. End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes

    SciTech Connect (OSTI)

    Rizk, Mazen; Antranikian, Garabed; Elleuche, Skander

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Multifunctional enzymes offer an interesting approach for biomass degradation. Black-Right-Pointing-Pointer Size and conformation of separate constructs play a role in the effectiveness of chimeras. Black-Right-Pointing-Pointer A connecting linker allows for maximal flexibility and increased thermostability. Black-Right-Pointing-Pointer Genes with functional similarities are the best choice for fusion candidates. -- Abstract: The reduction of fossil fuels, coupled with its increase in price, has made the search for alternative energy resources more plausible. One of the topics gaining fast interest is the utilization of lignocellulose, the main component of plants. Its primary constituents, cellulose and hemicellulose, can be degraded by a series of enzymes present in microorganisms, into simple sugars, later used for bioethanol production. Thermophilic bacteria have proven to be an interesting source of enzymes required for hydrolysis since they can withstand high and denaturing temperatures, which are usually required for processes involving biomass degradation. However, the cost associated with the whole enzymatic process is staggering. A solution for cost effective and highly active production is through the construction of multifunctional enzyme complexes harboring the function of more than one enzyme needed for the hydrolysis process. There are various strategies for the degradation of complex biomass ranging from the regulation of the enzymes involved, to cellulosomes, and proteins harboring more than one enzymatic activity. In this review, the construction of multifunctional biomass degrading enzymes through end-to-end gene fusions, and its impact on production and activity by choosing the enzymes and linkers is assessed.

  8. Anaerobic oxidation of short-chain alkanes in hydrothermal sediments: potential influences on sulfur cycling and microbial diversity

    SciTech Connect (OSTI)

    Adams, MM; Hoarfrost, AL; Bose, A; Joye, SB; Girguis, PR

    2013-05-14

    Short-chain alkanes play a substantial role in carbon and sulfur cycling at hydrocarbon-rich environments globally, yet few studies have examined the metabolism of ethane (C-2), propane (C-3), and butane (C-4) in anoxic sediments in contrast to methane (C-1). In hydrothermal vent systems, short-chain alkanes are formed over relatively short geological time scales via thermogenic processes and often exist at high concentrations. The sediment-covered hydrothermal vent systems at Middle Valley (MV Juan de Fuca Ridge) are an ideal site for investigating the anaerobic oxidation of C-1-C-4 alkanes, given the elevated temperatures and dissolved hydrocarbon species characteristic of these metalliferous sediments. We examined whether MV microbial communities oxidized C-1-C-4 alkanes under mesophilic to thermophilic sulfate-reducing conditions. Here we present data from discrete temperature (25, 55, and 75 degrees C) anaerobic batch reactor incubations of MV sediments supplemented with individual alkanes. Co-registered alkane consumption and sulfate reduction (SR) measurements provide clear evidence for C-1-C-4 alkane oxidation linked to SR over time and across temperatures. In these anaerobic batch reactor sediments, 16S ribosomal RNA pyrosequencing revealed that Deltaproteobacteria, particularly a novel sulfate-reducing lineage, were the likely phylotypes mediating the oxidation of C-2-C-4 alkanes. Maximum C-1-C-4 alkane oxidation rates occurred at 55 degrees C, which reflects the mid-core sediment temperature profile and corroborates previous studies of rate maxima for the anaerobic oxidation of methane (AOM). Of the alkanes investigated, C-3 was oxidized at the highest rate over time, then C-4, C-2, and C-1, respectively. The implications of these results are discussed with respect to the potential competition between the anaerobic oxidation of C-2-C(4)alkanes with AOM for available oxidants and the influence on the fate of C-1 derived from these hydrothermal systems.

  9. Fair Oaks Dairy Farms Cellulosic Ethanol Technology Review Summary

    SciTech Connect (OSTI)

    Andrew Wold; Robert Divers

    2011-06-23

    At Fair Oaks Dairy, dried manure solids (''DMS'') are currently used as a low value compost. United Power was engaged to evaluate the feasibility of processing these DMS into ethanol utilizing commercially available cellulosic biofuels conversion platforms. The Fair Oaks Dairy group is transitioning their traditional ''manure to methane'' mesophilic anaerobic digester platform to an integrated bio-refinery centered upon thermophilic digestion. Presently, the Digested Manure Solids (DMS) are used as a low value soil amendment (compost). United Power evaluated the feasibility of processing DMS into higher value ethanol utilizing commercially available cellulosic biofuels conversion platforms. DMS was analyzed and over 100 potential technology providers were reviewed and evaluated. DMS contains enough carbon to be suitable as a biomass feedstock for conversion into ethanol by gasification technology, or as part of a conversion process that would include combined heat and power. In the first process, 100% of the feedstock is converted into ethanol. In the second process, the feedstock is combusted to provide heat to generate electrical power supporting other processes. Of the 100 technology vendors evaluated, a short list of nine technology providers was developed. From this, two vendors were selected as finalists (one was an enzymatic platform and one was a gasification platform). Their selection was based upon the technical feasibility of their systems, engineering expertise, experience in commercial or pilot scale operations, the ability or willingness to integrate the system into the Fair Oaks Biorefinery, the know-how or experience in producing bio-ethanol, and a clear path to commercial development.

  10. Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

    SciTech Connect (OSTI)

    Makarova, Kira S.; Omelchenko, Marina V.; Gaidamakova, Elena K.; Matrosova, Vera Y.; Vasilenko, Alexander; Zhai, Min; Lapidus, Alla; Copeland, Alex; Kim, Edwin; Land, Miriam; Mavrommatis, Konstantinos; Pitluck, Samuel; Richardson, Paul M.; Detter, Chris; Brettin, Thomas; Saunders, Elizabeth; Lai, Barry; Ravel, Bruce; Kemner, Kenneth M.; Wolf, Yuri I.; Sorokin, Alexander; Gerasimova, Anna V.; Gelfand, Mikhail S.; Fredrickson, James K.; Koonin, Eugene V.; Daly, Michael J.

    2007-07-24

    Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at itsoptimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.

  11. The Genome Sequence of the psychrophilic archaeon, Methanococcoides burtonii: the Role of Genome Evolution in Cold-adaptation

    SciTech Connect (OSTI)

    Allen, Michelle A.; Lauro, Federico M.; Williams, Timothy J.; Burg, Dominic; Siddiqui, Khawar S.; De Francisci, David; Chong, Kevin W.Y.; Pilak, Oliver; Chew, Hwee H.; De Maere, Matthew Z.; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R.; Galperin, Michael Y.; Anderson, Iain J.; Ivanova, Natalia; Dalin, Eileen; Martinez, Michelle; Lapidus, Alla; Hauser, Loren; Land, Miriam; Thomas, Torsten; Cavicchioli, Ricardo

    2009-04-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- and {delta}-proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they play an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the last several thousand years as it adapted from a marine, to an Antarctic lake environment.

  12. Anaerobic microbial dissolution of lead and production of organic acids

    DOE Patents [OSTI]

    Francis, A.J.; Dodge, C.; Chendrayan, K.

    1986-02-28

    The present invention relates to a method of solubilizing lead, in the form of lead oxide, found in industrial wastes, before these wastes are dumped into the environment. The lead is solubilized by dissolving the lead oxide in the wastes through contact with an anaerobic bacterial culture containing the bacterium ATCC No. 53464. The solubilized lead can then be removed from the wastes by chemical separation. It could also be removed by extending the contact period with the bacterial culture. As the culture grows, the solubilized lead is removed from the wastes by bioaccumulation by the microorganism or by immobilization by a polymer-like material produced by the microorganism. At this point, the lead is then removed from the wastes when the waste material is separated from the bacterial culture. If desired, the bacterial culture could be digested at this point to yield relatively pure lead for further industrial use.

  13. Nucleic acid compositions and the encoding proteins

    DOE Patents [OSTI]

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  14. Crystal Structure of AGR_C_4470p from Agrobacterium tumefaciens

    SciTech Connect (OSTI)

    Vorobiev,S.; Neely, H.; Seetharaman, J.; Ma, L.; Xiao, R.; Acton, T.; Montelione, G.; Tong, L.

    2007-01-01

    We report here the crystal structure at 2.0 {angstrom} resolution of the AGR{_}C{_}4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR{_}C{_}4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR{_}C{_}4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR{_}C{_}4470p in E. coli, in addition to the ChuS protein.

  15. Nucleic acids, compositions and uses thereof

    DOE Patents [OSTI]

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2012-02-21

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  16. Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

    SciTech Connect (OSTI)

    Hawwa, Renda; Aikens, John; Turner, Robert J.; Santarsiero, Bernard D.; Mescar, Andrew D.

    2009-08-31

    A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

  17. Engineered plant biomass particles coated with biological agents

    DOE Patents [OSTI]

    Dooley, James H.; Lanning, David N.

    2014-06-24

    Plant biomass particles coated with a biological agent such as a bacterium or seed, characterized by a length dimension (L) aligned substantially parallel to a grain direction and defining a substantially uniform distance along the grain, a width dimension (W) normal to L and aligned cross grain, and a height dimension (H) normal to W and L. In particular, the L.times.H dimensions define a pair of substantially parallel side surfaces characterized by substantially intact longitudinally arrayed fibers, the W.times.H dimensions define a pair of substantially parallel end surfaces characterized by crosscut fibers and end checking between fibers, and the L.times.W dimensions define a pair of substantially parallel top and bottom surfaces.

  18. On-line monitoring of aerobic bioremediation with bioluminescent reporter microbes. Final report, July 1991--December 1994

    SciTech Connect (OSTI)

    Sayler, G.S.

    1995-03-01

    A critical issue in the biological characterization of contaminated sites and in the evaluation of relative bioremediation treatment efficiencies is the development of appropriate monitoring methods for the assessment of pollutant bioavailability and microbial in situ activity potential. In nature, pollutants are found dispersed among the solid, liquid and gaseous phases of the complex environments rendering the analytical estimation of their bioavailability and degradation more difficult and irrelevant. Ex situ and extractive analytical techniques have only been misrepresentative of the natural conditions and often resulted in inaccurate estimates of pollutants mass transfer. In this project, the bioluminescent bioreporter bacterium P. Fluorescens HK44 was integrated to an optical device, capable of conducting emitted light, and used as an online biosensor of naphthalene and salicylate. The physiological requirements of the bacteria and the physical limitations of the biosensor were also determined.

  19. Complete genome sequence of Eggerthella lenta type strain (IPP VPI 0255T)

    SciTech Connect (OSTI)

    Saunders, Elizabeth H; Pukall, Rudiger; Birte, Abt; Lapidus, Alla L.; Glavina Del Rio, Tijana; Copeland, A; Tice, Hope; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; Bruce, David; Goodwin, Lynne A.; Pitluck, Sam; Ivanova, N; Mavromatis, K; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Hauser, Loren John; Chang, Yun-Juan; Jeffries, Cynthia; Chain, Patrick S. G.; Meincke, Linda; Sims, David; Brettin, Tom; Detter, J. Chris; Goker, Markus; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Han, Cliff

    2009-01-01

    Eggerthella lenta (Eggerth 1935) Wade et al. 1999, emended W rdemann et al. 2009 is the type species of the genus Eggerthella, which belongs to the actinobacterial family Coriobacteriaceae. E. lenta is a Gram-positive, non-motile, non-sporulating pathogenic bacterium that can cause severe bacteremia. The strain described in this study has been isolated from a rectal tumor in 1935. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the genus Eggerthella, and the 3,632,260 bp long single replicon genome with its 3123 protein-coding and 58 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  20. Complete genome sequence of Olsenella uli type strain (VPI D76D-27CT)

    SciTech Connect (OSTI)

    Goker, Markus; Held, Brittany; Lucas, Susan; Nolan, Matt; Yasawong, Montri; Glavina Del Rio, Tijana; Tice, Hope; Cheng, Jan-Fang; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Ivanova, N; Mavromatis, K; Mikhailova, Natalia; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Hauser, Loren John; Chang, Yun-Juan; Jeffries, Cynthia; Rohde, Manfred; Sikorski, Johannes; Pukall, Rudiger; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter

    2010-01-01

    Olsenella uli (Olsen et al. 1991) Dewhirst et al. 2001 is the type species of the genus Olsenella, which belongs to the actinobacterial family Coriobacteriaceae. The species is of interest because it is frequently isolated from dental plaque in periodontitis patients and can cause primary endodontic infection. The species is a Gram-positive, non-motile and non-sporulating bacterium. The strain described in this study has been isolated from human gingival crevices in 1982. This is the first completed sequence of the genus Olsenella and the fifth sequence from the family Coriobacteriaceae. The 2,051,896 bp long genome with its 1,795 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  1. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOE Patents [OSTI]

    Bavykin, Sergei G.; Mirzabekov, Andrei D.

    2007-10-30

    The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  2. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOE Patents [OSTI]

    Bavykin, Sergei G.; Mirzabekova, legal representative, Natalia V.; Mirzabekov, deceased, Andrei D.

    2007-12-04

    The present invention relates to methods and compositions for using nucleotide sequence variations of 16S and 23S rRNA within the B. cereus group to discriminate a highly infectious bacterium B. anthracis from closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations and discriminate B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed samples, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  3. Microbial production of multi-carbon chemicals and fuels from water and carbon dioxide using electric current

    DOE Patents [OSTI]

    Lovley, Derek R; Nevin, Kelly

    2015-11-03

    The invention provides systems and methods for generating organic compounds using carbon dioxide as a source of carbon and electrical current as an energy source. In one embodiment, a reaction cell is provided having a cathode electrode and an anode electrode that are connected to a source of electrical power, and which are separated by a permeable membrane. A biological film is provided on the cathode. The biological film comprises a bacterium that can accept electrons and that can convert carbon dioxide to a carbon-bearing compound and water in a cathode half-reaction. At the anode, water is decomposed to free molecular oxygen and solvated protons in an anode half-reaction. The half-reactions are driven by the application of electrical current from an external source. Compounds that have been produced include acetate, butanol, 2-oxobutyrate, propanol, ethanol, and formate.

  4. Life Redefined: Microbes Built with Arsenic

    SciTech Connect (OSTI)

    Webb, Sam

    2011-03-22

    Life can survive in many harsh environments, from extreme heat to the presence of deadly chemicals. However, life as we know it has always been based on the same six elements -- carbon, oxygen, nitrogen, hydrogen, sulfur and phosphorus. Now it appears that even this rule has an exception. In the saline and poisonous environment of Mono Lake, researchers have found a bacterium that can grow by incorporating arsenic into its structure in place of phosphorus. X-ray images taken at SLAC's synchrotron light source reveal that this microbe may even use arsenic as a building block for DNA. Please join us as we describe this discovery, which rewrites the textbook description of how living cells work.

  5. Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

    SciTech Connect (OSTI)

    Puri, AW; Owen, S; Chu, F; Chavkin, T; Beck, DAC; Kalyuzhnaya, MG; Lidstrom, ME

    2015-02-10

    Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.

  6. Novel Biological Conversion of Hydrogen and Carbon Dioxide Directly into Biodiesel: Cooperative Research and Development Final Report, CRADA Number: CRD-10-408

    SciTech Connect (OSTI)

    Maness, P. C.

    2014-06-01

    OPX Biotechnologies, Inc. (OPX), the National Renewable Energy Laboratory (NREL), and Johnson Matthey will develop and optimize a novel, engineered microorganism that directly produces biodiesel from renewable hydrogen (H2) and carbon dioxide (CO2). The proposed process will fix CO2 utilizing H2 to generate an infrastructure-compatible, energy-dense fuel at costs of less than $2.50 per gallon, with water being produced as the primary byproduct. NREL will perform metabolic engineering on the bacterium Cupriavidus necator (formerly Ralstonia eutropha) and a techno-economic analysis to guide future scale-up work. H2 and CO2 uptakes rates will be genetically increased, production of free fatty acids will be enhanced and their degradation pathway blocked in order to meet the ultimate program goals.

  7. Microbial engineering of nano-heterostructures; biological synthesis of a magnetically-recoverable palladium nanocatalyst

    SciTech Connect (OSTI)

    Coker, V. S.; Bennett, J. A.; Telling, N.; Charnock, J. M.; van der Laan, G.; Pattrick, R. A. D.; Pearce, C. I; Cutting, R. S.; Shannon, I. J.; Wood, J.; Arenholz, E.; Vaughan, D. J.; Lloyd, J. R.

    2009-12-01

    Precious metals supported on ferrimagnetic particles form a diverse range of catalysts. Here we show a novel biotechnological route for the synthesis of a heterogeneous catalyst consisting of reactive palladium nanoparticles arrayed on a biomagnetite support. The magnetic support was synthesized at ambient temperature by the Fe(III)-reducing bacterium, Geobacter sulfurreducens, and facilitated ease of recovery of the catalyst with superior performance due to reduced agglomeration. Arrays of palladium nanoparticles were deposited on the nanomagnetite using a simple one-step method without the need to modify the biomineral surface most likely due to an organic coating priming the surface for Pd adsorption. A combination of EXAFS and XPS showed the particles to be predominantly metallic in nature. The Pd{sup 0}-biomagnetite was tested for catalytic activity in the Heck Reaction coupling iodobenzene to ethyl acrylate or styrene and near complete conversion to ethyl cinnamate or stilbene was achieved within 90 and 180 min, respectively.

  8. Small Talk: Cell-to-Cell Communication in Bacteria

    ScienceCinema (OSTI)

    Bassler, Bonnie [Princeton University, Princeton, New Jersey, United States

    2010-01-08

    Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.

  9. Complete genome sequence of Catenulispora acidiphila type strain (ID 139908T)

    SciTech Connect (OSTI)

    Copeland, Alex; Lapidus, Alla; Rio, Tijana GlavinaDel; Nolan, Matt; Lucas, Susan; Chen, Feng; Tice, Hope; Cheng, Jan-Fang; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Mikhailova, Natalia; Pati, Amrita; Ivanova, Natalia; Mavromatis, Konstantinos; Chen, Amy; Palaniappan, Krishna; Chain, Patrick; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C.; Chertkov, Olga; Brettin, Thomas; Detter, John C.; Han, Cliff; Ali, Zahid; Tindall, Brian J.; Goker, Markus; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter

    2009-05-20

    Catenulispora acidiphila Busti et al. 2006 is the type species of the genus Catenulispora, and is of interest because of the rather isolated phylogenetic location of the genomically little studied suborder Catenulisporineae within the order Actinomycetales. C. acidiphilia is known for its acidophilic, aerobic lifestyle, but can also grow scantly under anaerobic conditions. Under regular conditions C. acidiphilia grows in long filaments of relatively short aerial hyphae with marked septation. It is a free living, non motile, Gram-positive bacterium isolated from a forest soil sample taken from a wooded area in Gerenzano, Italy. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the actinobacterial family Catenulisporaceae, and the 10,467,782 bp long single replicon genome with its 9056 protein-coding and 69 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  10. Complete genome sequence of Beutenbergia cavernae type strain (HKI 0122T)

    SciTech Connect (OSTI)

    Land, Miriam; Pukall, Rudiger; Abt, Birte; Goker, Markus; Rohde, Manfred; Glavina Del Rio, Tijana; Tice, Hope; Copeland, Alex; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavrommatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C.; Saunders, Elizabeth; Brettin, Thomas; Detter, John C.; Han, Cliff; Chain, Patrick; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter; Lapidus, Alla

    2009-05-20

    Beutenbergia cavernae (Groth et al. 1999) is the type species of the genus and is of phylogenetic interest because of its isolated location in the actinobacterial suborder Micrococcineae. B. cavernae HKI 0122T is a Gram-positive, non-motile, non-spore-forming bacterium isolated from a cave in Guangxi (China). B. cavernae grows best under aerobic conditions and shows a rod-coccus growth cycle. Its cell wall peptidoglycan contains the diagnostic L-lysine - L-glutamate interpeptide bridge. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the poorly populated micrococcineal family Beutenbergiaceae, and this 4,669,183 bp long single replicon genome with its 4225 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  11. Complete genome sequence of Brachybacterium faecium type strain (Schefferle 6-10T)

    SciTech Connect (OSTI)

    Lapidus, Alla; Pukall, Rudiger; LaButti, Kurt; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Chen, Feng; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Rohde, Manfred; Goker, Markus; Pati, Amrita; Ivanova, Natalia; Mavrommatis, Konstantinos; Chen, Amy; Palaniappan, Krishna; D'haeseleer, Patrik; Chain, Patrick; Bristow, Jim; Eisen, Johnathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter

    2009-05-20

    Brachybacterium faecium Collins et al. 1988 is the type species of the genus, and is of phylogenetic interest because of its location in the Dermabacteraceae, a rather isolated family within the actinobacterial suborder Micrococcineae. B. faecium is known for its rod-coccus growth cycle and the ability to degrade uric acid. It grows aerobically or weakly anaerobically. The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, originally isolated from poultry deep litter. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the actinobacterial family Dermabacteraceae, and the 3,614,992 bp long single replicon genome with its 3129 protein-coding and 69 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  12. Inducible error-prone repair in B. subtilis. Final report, September 1, 1979-June 30, 1981

    SciTech Connect (OSTI)

    Yasbin, R. E.

    1981-06-01

    The research performed under this contract has been concentrated on the relationship between inducible DNA repair systems, mutagenesis and the competent state in the gram positive bacterium Bacillus subtilis. The following results have been obtained from this research: (1) competent Bacillus subtilis cells have been developed into a sensitive tester system for carcinogens; (2) competent B. subtilis cells have an efficient excision-repair system, however, this system will not function on bacteriophage DNA taken into the cell via the process of transfection; (3) DNA polymerase III is essential in the mechanism of the process of W-reactivation; (4) B. subtilis strains cured of their defective prophages have been isolated and are now being developed for gene cloning systems; (5) protoplasts of B. subtilis have been shown capable of acquiring DNA repair enzymes (i.e., enzyme therapy); and (6) a plasmid was characterized which enhanced inducible error-prone repair in a gram positive organism.

  13. PHOTOBIOLOGICAL HYDROGEN RESEARCH

    SciTech Connect (OSTI)

    Philippidis, George; Tek, Vekalet

    2009-07-01

    The project objectives are to develop bio-hydrogen production by:  Cloning the structural and subunit genes (cooKMUX and cooLH resp.) of the O{sub 2}- tolerant NiFe-hydrogenase from the photosynthetic bacterium Rubrivivax gelatinosus CBS strain in collaboration with NREL.  Cloning the active site maturation genes (hypA-F) of the CBS hydrogenase in collaboration with NREL.  Transforming the structural and subunits genes, along with the maturation genes, into E. coli and determining the minimum number of genes required for expression of a functional hydrogenase.  Upon expression of a functional hydrogenase, purifying and characterizing the recombinant hydrogenase from E. coli and performing bioreactor studies to optimize hydrogen production by E. coli.

  14. Recombinant glucose uptake system

    DOE Patents [OSTI]

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  15. Complete genome sequence of Coraliomargarita akajimensis type strain (04OKA010-24T)

    SciTech Connect (OSTI)

    Mavromatis, Konstantinos; Abt, Birte; Brambilla, Evelyne; Lapidus, Alla; Copeland, Alex; Desphande, Shweta; Nolan, Matt; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Han, Cliff; Detter, John C.; Woyke, Tanja; Goodwin, Lynne; Pitluck, Sam; Held, Brittany; Brettin, Thomas; Tapia, Roxanne; Ivanova, Natalia; Mikhailova, Natalia; Pati, Amrita; Liolios, Konstantinos; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D.; Rohde, Manfred; Gö ker, Markus; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C.

    2010-06-25

    Coraliomargarita akajimensis Yoon et al. 2007 the type species of the genus Coraliomargarita. C. akajimensis is an obligately aerobic, Gram-negative, non-spore-forming, non-motile, spherical bacterium which was isolated from seawater surrounding the hard coral Galaxea fascicularis. C. akajimensis organism is of special interest because of its phylogenetic position in a genomically purely studied area in the bacterial diversity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Puniceicoccaceae. The 3,750,771 bp long genome with its 3,137 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  16. Alteration of Iron-rich Lacustrine Sediments by Dissimilatory Iron-reducing Bacteria

    SciTech Connect (OSTI)

    Crowe,S.; O'Niell, A.; Kulezycki, E.; Weisener, C.; Roberts, J.; Fowle, D.

    2007-01-01

    The reactivity of trace elements in lake sediments towards microbial metal reduction was evaluated using spectroscopy, chemical extractions and incubations in a minimal media with the DIR bacterium Shewanella putrefaciens 200R. Micro-XRF measurements demonstrated the association of Cr, and Ni with Mn-rich phases. The onset of anaerobic conditions resulted in the rapid release of trace metals (Cr, Ni, Co) from the sediments with the progressive dissolution of a reactive Mn component. This fraction was approximately equivalent to that liberated by chemical extractions designed to operationally select for Mn phases. These results suggest that studies aiming to assess metal dissolution in anaerobic soils and sediments should attempt to discriminate between metals associated with Mn and Fe (hydr)oxides, the former being more reactive and likely dissolved to a greater extent.

  17. Yellow affinity substance involved in the cellulolytic system of Clostridium thermocellum

    SciTech Connect (OSTI)

    Ljungdahl, L.G.; Pettersson, B.; Eriksson, K.E.; Wiegel, J.

    1983-01-01

    Clostridium thermocellum produces a yellow substance when fermenting cellulose. This substance is attached to the cellulose particles. Cellulose with the yellow substance, obtained from cultures of C. thermocellum, binds effectively endo-1,4-..beta..-glucanase produced by the bacterium and was used in an affinity column for purification of the enzyme. At the beginning of fermentation of cellulose, most of the endoglucanase was bound to the yellow cellulose. As the fermentation proceeded, the enzyme appeared free in the culture fluid. The endoglucanase bound to the yellow cellulose could be extracted by distilled water from the cellulose, but not by solutions with 5 mM or higher concentrations of salts or buffers. It is proposed that the yellow substance is involved in the cellulolytic system of C. thermocellum. 30 references, 3 figures, 3 tables.

  18. Structure of cellobiose phosphorylase from Clostridium thermocellum in complex with phosphate

    SciTech Connect (OSTI)

    Bianchetti, Christopher M.; Elsen, Nathaniel L.; Fox, Brian G.; Phillips, Jr., George N.

    2012-03-27

    Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into -D-glucose 1-phosphate and D-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-terminal domain, helical linker, (/)6-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 {angstrom} resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination.

  19. Identification of metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii

    SciTech Connect (OSTI)

    Miquel, Sylvie; Leclerc, Marion; Martin, Rebeca; Chain, Florian; Lenoir, Marion; Raguideau, Sébastien; Hudault, Sylvie; Bridonneau, Chantal; Northen, Trent; Bowen, Benjamin; Bermúdez-Humarán, Luis G.; Sokol, Harry; Thomas, Muriel; Langella, Philippe

    2015-04-21

    Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable and stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood.

  20. Quantitative Tools for Dissection of Hydrogen-Producing Metabolic Networks-Final Report

    SciTech Connect (OSTI)

    Rabinowitz, Joshua D.; Dismukes, G.Charles.; Rabitz, Herschel A.; Amador-Noguez, Daniel

    2012-10-19

    During this project we have pioneered the development of integrated experimental-computational technologies for the quantitative dissection of metabolism in hydrogen and biofuel producing microorganisms (i.e. C. acetobutylicum and various cyanobacteria species). The application of these new methodologies resulted in many significant advances in the understanding of the metabolic networks and metabolism of these organisms, and has provided new strategies to enhance their hydrogen or biofuel producing capabilities. As an example, using mass spectrometry, isotope tracers, and quantitative flux-modeling we mapped the metabolic network structure in C. acetobutylicum. This resulted in a comprehensive and quantitative understanding of central carbon metabolism that could not have been obtained using genomic data alone. We discovered that biofuel production in this bacterium, which only occurs during stationary phase, requires a global remodeling of central metabolism (involving large changes in metabolite concentrations and fluxes) that has the effect of redirecting resources (carbon and reducing power) from biomass production into solvent production. This new holistic, quantitative understanding of metabolism is now being used as the basis for metabolic engineering strategies to improve solvent production in this bacterium. In another example, making use of newly developed technologies for monitoring hydrogen and NAD(P)H levels in vivo, we dissected the metabolic pathways for photobiological hydrogen production by cyanobacteria Cyanothece sp. This investigation led to the identification of multiple targets for improving hydrogen production. Importantly, the quantitative tools and approaches that we have developed are broadly applicable and we are now using them to investigate other important biofuel producers, such as cellulolytic bacteria.

  1. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    SciTech Connect (OSTI)

    Li, Yongchao; Tschaplinski, Timothy J; Engle, Nancy L; Hamilton, Choo Yieng; Rodriguez, Jr., Miguel; Liao, James C; Schadt, Christopher Warren; Guss, Adam M; Yang, Yunfeng; Graham, David E

    2012-01-01

    Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from complex biomass substrates.

  2. Identification of metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Miquel, Sylvie; Leclerc, Marion; Martin, Rebeca; Chain, Florian; Lenoir, Marion; Raguideau, Sébastien; Hudault, Sylvie; Bridonneau, Chantal; Northen, Trent; Bowen, Benjamin; et al

    2015-04-21

    Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified on the basis of human clinical data. The mechanisms underlying its beneficial effects are still unknown. Gnotobiotic mice harboring F. prausnitzii (A2-165) and Escherichia coli (K-12 JM105) were subjected to 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis. The inflammatory colitis scores and a gas chromatography-time of flight (GC/TOF) mass spectrometry-based metabolomic profile were monitored in blood, ileum, cecum, colon, and feces in gnotobiotic mice. The potential anti-inflammatory metabolites were tested in vitro. We obtained stable E. coli and F. prausnitzii-diassociated mice in which E. coli primed the gastrointestinal tract (GIT), allowing a durable andmore » stable establishment of F. prausnitzii. The disease activity index, histological scores, myeloperoxidase (MPO) activity, and serum cytokine levels were significantly lower in the presence of F. prausnitzii after TNBS challenge. The protective effect of F. prausnitzii against colitis was correlated to its implantation level and was linked to overrepresented metabolites along the GIT and in serum. Among 983 metabolites in GIT samples and serum, 279 were assigned to known chemical reactions. Some of them, belonging to the ammonia (α-ketoglutarate), osmoprotective (raffinose), and phenolic (including anti-inflammatory shikimic and salicylic acids) pathways, were associated with a protective effect of F. prausnitzii, and the functional link was established in vitro for salicylic acid. We show for the first time that F. prausnitzii is a highly active commensal bacterium involved in reduction of colitis through in vivo modulation of metabolites along the GIT and in the peripheral blood.« less

  3. Nonphotochemical Hole-Burning Studies of Energy Transfer Dynamics in Antenna Complexes of Photosynthetic Bacteria

    SciTech Connect (OSTI)

    Satoshi Matsuzaki

    2002-06-27

    This thesis contains the candidate's original work on excitonic structure and energy transfer dynamics of two bacterial antenna complexes as studied using spectral hole-burning spectroscopy. The general introduction is divided into two chapters (1 and 2). Chapter 1 provides background material on photosynthesis and bacterial antenna complexes with emphasis on the two bacterial antenna systems related to the thesis research. Chapter 2 reviews the underlying principles and mechanism of persistent nonphotochemical hole-burning (NPHB) spectroscopy. Relevant energy transfer theories are also discussed. Chapters 3 and 4 are papers by the candidate that have been published. Chapter 3 describes the application of NPHB spectroscopy to the Fenna-Matthews-Olson (FMO) complex from the green sulfur bacterium Prosthecochloris aestuarii; emphasis is on determination of the low energy vibrational structure that is important for understanding the energy transfer process associated within three lowest energy Q{sub y}-states of the complex. The results are compared with those obtained earlier on the FMO complex from Chlorobium tepidum. In Chapter 4, the energy transfer dynamics of the B800 molecules of intact LH2 and B800-deficient LH2 complexes of the purple bacterium Rhodopseudomonas acidophila are compared. New insights on the additional decay channel of the B800 ring of bacteriochlorophyll{sub a} (BChl{sub a}) molecules are provided. General conclusions are given in Chapter 5. A version of the hole spectrum simulation program written by the candidate for the FMO complex study (Chapter 3) is included as an appendix. The references for each chapter are given at the end of each chapter.

  4. Continuous high-solids anaerobic co-digestion of organic solid wastes under mesophilic conditions

    SciTech Connect (OSTI)

    Kim, Dong-Hoon; Oh, Sae-Eun

    2011-09-15

    Highlights: > High-solids (dry) anaerobic digestion is attracting a lot of attention these days. > One reactor was fed with food waste (FW) and paper waste. > Maximum biogas production rate of 5.0 m{sup 3}/m{sup 3}/d was achieved at HRT 40 d and 40% TS. > The other reactor was fed with FW and livestock waste (LW). > Until a 40% LW content increase, the reactor exhibited a stable performance. - Abstract: With increasing concerns over the limited capacity of landfills, conservation of resources, and reduction of CO{sub 2} emissions, high-solids (dry) anaerobic digestion of organic solid waste (OSW) is attracting a great deal of attention these days. In the present work, two dry anaerobic co-digestion systems fed with different mixtures of OSW were continuously operated under mesophilic conditions. Dewatered sludge cake was used as a main seeding source. In reactor (I), which was fed with food waste (FW) and paper waste (PW), hydraulic retention time (HRT) and solid content were controlled to find the maximum treatability. At a fixed solid content of 30% total solids (TS), stable performance was maintained up to an HRT decrease to 40 d. However, the stable performance was not sustained at 30 d HRT, and hence, HRT was increased to 40 d again. In further operation, instead of decreasing HRT, solid content was increased to 40% TS, which was found to be a better option to increase the treatability. The biogas production rate (BPR), CH{sub 4} production yield (MPY) and VS reduction achieved in this condition were 5.0 m{sup 3}/m{sup 3}/d, 0.25 m{sup 3} CH{sub 4}/g COD{sub added}, and 80%, respectively. Reactor (II) was fed with FW and livestock waste (LW), and LW content was increased during the operation. Until a 40% LW content increase, reactor (II) exhibited a stable performance. A BPR of 1.7 m{sup 3}/m{sup 3}/d, MPY of 0.26 m{sup 3} CH{sub 4}/g COD{sub added}, and VS reduction of 72% was achieved at 40% LW content. However, when the LW content was increased to 60%, there was a significant performance drop, which was attributed to free ammonia inhibition. The performances in these two reactors were comparable to the ones achieved in the conventional wet digestion and thermophilic dry digestion processes.

  5. A nanostructure-initiator mass spectrometry-based enzyme activity assay

    SciTech Connect (OSTI)

    Siuzdak, Gary; Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren; Yannone, Steven M.; Wong, Chi-Huey; Siuzdak, Gary

    2008-03-10

    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing {beta}-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 C and 5.5, respectively, and the activity was inhibited by both phenylethyl-{beta}-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced {gamma}-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis. The interest in leveraging mass spectrometry for studying enzyme activities in complex biological samples derives from its high sensitivity and specificity; however, signal suppression and significant sample preparation requirements limit its overall utility (1). Here we describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay, which uses the fluorous liquid-coated surface of NIMS (2) to noncovalently attach enzyme substrates by means of fluorous tags. Enzymes play essential roles in a wide range of cellular processes and account for >20% of all drug targets (3). In addition, enzymes have found great utility in organic synthesis because they can efficiently catalyze chemical transformations that are difficult and inefficient to catalyze using conventional synthetic approaches. Furthermore, enzymatic transformations are particularly useful in reactions requiring multiple functional groups or stereo/regiochemically defined products (4). These properties make them particularly well suited for the synthesis and degradation of carbohydrates (5). Indeed, enzymatic approaches have found widespread applications in glycobiology (6, 7) and are of intense interest for the utilization of plant biomass for biofuels (8).

  6. Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass

    SciTech Connect (OSTI)

    Wilson, Charlotte M; Rodriguez Jr, Miguel; Johnson, Courtney M; Martin, S L.; Chu, Tzu Ming; Wolfinger, Russ; Hauser, Loren John; Land, Miriam L; Klingeman, Dawn Marie; Tschaplinski, Timothy J; Mielenz, Jonathan R; Brown, Steven D

    2013-01-01

    Background The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms. Results C. thermocellum ATCC 27405 fermentations were conducted with a 5 g/L solid substrate loading of either pretreated switchgrass or Populus. Quantitative saccharification and inductively coupled plasma emission spectroscopy (ICP-ES) for elemental analysis revealed composition differences between biomass substrates, which may have influenced growth and transcriptomic profiles. High quality RNA was prepared for C. thermocellum grown on solid substrates and transcriptome profiles were obtained for two time points during active growth (12 hours and 37 hours postinoculation). A comparison of two transcriptomic analytical techniques, microarray and RNA-seq, was performed and the data analyzed for statistical significance. Large expression differences for cellulosomal genes were not observed. We updated gene predictions for the strain and a small novel gene, Cthe_3383, with a putative AgrD peptide quorum sensing function was among the most highly expressed genes. RNAseq data also supported different small regulatory RNA predictions over others. The DNA microarray gave a greater number (2,351) of significant genes relative to RNA-seq (280 genes when normalized by the kernel density mean of M component (KDMM) method) in an analysis of variance (ANOVA) testing method with a 5 % false discovery rate (FDR). When a 2-fold difference in expression threshold was applied, 73 genes were significantly differentially expressed in common between the two techniques. Sulfate and phosphate uptake/utilization genes, along with genes for a putative efflux pump system were some of the most differentially regulated transcripts when profiles for C. thermocellum grown on either pretreated switchgrass or Populus were compared. Conclusions Our results suggest that a high degree of agreement in differential gene expression measurements between transcriptomic platforms is possible, but choosing an appropriate normalization regime is essential.

  7. Clostridium thermocellum Transcriptomic Profiles after Exposure to Furfural or Heat Stress

    SciTech Connect (OSTI)

    Wilson, Charlotte M; Yang, Shihui; Rodriguez, Jr., Miguel; Ma, Qin; Johnson, Courtney M; Dice, Lezlee T; Xu, Ying; Brown, Steven D

    2013-01-01

    Background The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP)biocatalyst for cellulosic ethanol production. It is capable of both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. A previous C. thermocellum ethanol stress study showed that largest transcriptomic response was in genes and proteins related to nitrogen uptake and metabolism. Results In this study, C. thermocellum was grown to mid-exponential phase and treated with furfural or heat to a final concentration of 3 g.L-1 or 68 C respectively to investigate general and specific physiological and regulatory stress responses. Samples were taken at 10, 30, 60 and 120 min post-shock, and from untreated control fermentations, for transcriptomic analyses and fermentation product determinations and compared to a published dataset from an ethanol stress study. Urea uptake genes were induced following furfural stress, but not to the same extent as ethanol stress and transcription from these genes was largely unaffected by heat stress. The largest transcriptomic response to furfural stress was genes for sulfate transporter subunits and enzymes in the sulfate assimilatory pathway, although these genes were also affected late in the heat and ethanol stress responses. Lactate production was higher in furfural treated culture, although the lactate dehydrogenase gene was not differentially expressed under this condition. Other redox related genes such as a copy of the rex gene, a bifunctional acetaldehyde-CoA/alcohol dehydrogenase and adjacent genes did show lower expression after furfural stress compared to the control, heat and ethanol fermentation profiles. Heat stress induced expression from chaperone related genes and overlap was observed with the responses to the other stresses. This study suggests the involvement of C. thermocellum genes with functions in oxidative stress protection, electron transfer, detoxification, sulfur and nitrogen acquisition, and DNA repair mechanisms in its stress responses and the use of different regulatory networks to coordinate and control adaptation. Conclusions This study has identified C. thermocellum gene regulatory motifs and aspects of physiology and gene regulation for further study. The nexus between future systems biology studies and recently developed genetic tools for C. thermocellum offers the potential for more rapid strain development and for broader insights into this organism s physiology and regulation.

  8. A STUDY ON LEGIONELLA PNEUMOPHILA, WATER CHEMISTRY, AND ATMOSPHERIC CONDITIONS IN COOLING TOWERS AT THE SAVANNAH RIVER SITE

    SciTech Connect (OSTI)

    Smith, C.; Brigmon, R.

    2009-10-20

    Legionnaires disease is a pneumonia caused by the inhalation of the bacterium Legionella pneumophila. The majority of illnesses have been associated with cooling towers since these devices can harbor and disseminate the bacterium in the aerosolized mist generated by these systems. Historically, Savannah River Site (SRS) cooling towers have had occurrences of elevated levels of Legionella in all seasons of the year and in patterns that are difficult to predict. Since elevated Legionella in cooling tower water are a potential health concern a question has been raised as to the best control methodology. In this work we analyze available chemical, biological, and atmospheric data to determine the best method or key parameter for control. The SRS 4Q Industrial Hygiene Manual, 4Q-1203, 1 - G Cooling Tower Operation and the SRNL Legionella Sampling Program, states that 'Participation in the SRNL Legionella Sampling Program is MANDATORY for all operating cooling towers'. The resulting reports include L. pneumophila concentration information in cells/L. L. pneumophila concentrations >10{sup 7} cells/L are considered elevated and unsafe so action must be taken to reduce these densities. These remedial actions typically include increase biocide addition or 'shocking'. Sometimes additional actions are required if the problem persists including increase tower maintenance (e.g. cleaning). Evaluation of 14 SRS cooling towers, seven water quality parameters, and five Legionella serogroups over a three-plus year time frame demonstrated that cooling tower water Legionella densities varied widely though out this time period. In fact there was no one common consistent significant variable across all towers. The significant factors that did show up most frequently were related to suspended particulates, conductivity, pH, and dissolved oxygen, not chlorine or bromine as might be expected. Analyses of atmospheric data showed that there were more frequent significant elevated Legionella concentrations when the dew point temperature was high--a summertime occurrence. However, analysis of the three years of Legionella monitoring data of the 14 different SRS Cooling Towers demonstrated that elevated concentrations are observed at all temperatures and seasons. The objective of this study is to evaluate the ecology of L. pneumophila including serogroups and population densities, chemical, and atmospheric data, on cooling towers at SRS to determine whether relationships exist among water chemistry, and atmospheric conditions. The goal is to more fully understand the conditions which inhibit or encourage L. pneumophila growth and supply this data and associated recommendations to SRS Cooling Tower personnel for improved management of operation. Hopefully this information could then be used to help control L. pneumophila growth more effectively in SRS cooling tower water.

  9. SPINE: SParse eIgengene NEtwork linking gene expression clusters in Dehalococcoides mccartyi to perturbations in experimental conditions

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Mansfeldt, Cresten B.; Logsdon, Benjamin A.; Debs, Garrett E.; Richardson, Ruth E.; Mande, Shekhar C.

    2015-02-25

    We present a statistical model designed to identify the effect of experimental perturbations on the aggregate behavior of the transcriptome expressed by the bacterium Dehalococcoides mccartyi strain 195. Strains of Dehalococcoides are used in sub-surface bioremediation applications because they organohalorespire tetrachloroethene and trichloroethene (common chlorinated solvents that contaminate the environment) to non-toxic ethene. However, the biochemical mechanism of this process remains incompletely described. Additionally, the response of Dehalococcoides to stress-inducing conditions that may be encountered at field-sites is not well understood. The constructed statistical model captured the aggregate behavior of gene expression phenotypes by modeling the distinct eigengenes of 100more » transcript clusters, determining stable relationships among these clusters of gene transcripts with a sparse network-inference algorithm, and directly modeling the effect of changes in experimental conditions by constructing networks conditioned on the experimental state. Based on the model predictions, we discovered new response mechanisms for DMC, notably when the bacterium is exposed to solvent toxicity. The network identified a cluster containing thirteen gene transcripts directly connected to the solvent toxicity condition. Transcripts in this cluster include an iron-dependent regulator (DET0096-97) and a methylglyoxal synthase (DET0137). To validate these predictions, additional experiments were performed. Continuously fed cultures were exposed to saturating levels of tetrachloethene, thereby causing solvent toxicity, and transcripts that were predicted to be linked to solvent toxicity were monitored by quantitative reverse-transcription polymerase chain reaction. Twelve hours after being shocked with saturating levels of tetrachloroethene, the control transcripts (encoding for a key hydrogenase and the 16S rRNA) did not significantly change. By contrast, transcripts for DET0137 and DET0097 displayed a 46.8±11.5 and 14.6±9.3 fold up-regulation, respectively, supporting the model. This is the first study to identify transcripts in Dehalococcoides that potentially respond to tetrachloroethene solvent-toxicity conditions that may be encountered near contamination source zones in sub-surface environments.« less

  10. Direct Involvement of ombB, omaB and omcB Genes in Extracellular Reduction of Fe(III) by Geobacter sulfurreducens PCA

    SciTech Connect (OSTI)

    Liu, Yimo; Fredrickson, Jim K.; Zachara, John M.; Shi, Liang

    2015-10-01

    The tandem gene clusters orfR-ombB-omaB-omcB and orfS-ombC-omaC-omcC of the metal-reducing bacterium Geobacter sulfurreducens PCA are responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III)-citrate and ferrihydrite [a poorly crystalline Fe(III) oxide]. Each gene cluster encodes a putative transcriptional factor (OrfR/OrfS), a porin-like outer-membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (c-Cyt, OmaB/OmaC) and an outer-membrane c-Cyt (OmcB/OmcC). The individual roles of OmbB, OmaB and OmcB in extracellular reduction of Fe(III), however, have remained either uninvestigated or controversial. Here, we showed that replacements of ombB, omaB, omcB and ombB-omaB with an antibiotic gene in the presence of ombC-omaC-omcC had no impact on reduction of Fe(III)-citrate by G. sulfurreducens PCA. Disruption of ombB, omaB, omcB and ombB-omaB in the absence of ombC-omaC-omcC, however, severely impaired the bacterial ability to reduce Fe(III)-citrate as well as ferrihydrite. These results unequivocally demonstrate an overlapping role of ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III) reduction by G. sulfurreducens PCA. Involvement of both ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III) reduction reflects the importance of these trans-outer membrane protein complexes in the physiology of this bacterium. Moreover, the kinetics of Fe(III)-citrate and ferrihydrite reduction by these mutants in the absence of ombC-omaC-omcC were nearly identical, which clearly show that OmbB, OmaB and OmcB contribute equally to extracellular Fe(III) reduction. Finally, orfS was found to have a negative impact on the extracellular reduction of Fe(III)-citrate and ferrihydrite in G. sulfurreducens PCA probably by serving as a transcriptional repressor.

  11. SPINE: SParse eIgengene NEtwork linking gene expression clusters in Dehalococcoides mccartyi to perturbations in experimental conditions

    SciTech Connect (OSTI)

    Mansfeldt, Cresten B.; Logsdon, Benjamin A.; Debs, Garrett E.; Richardson, Ruth E.; Mande, Shekhar C.

    2015-02-25

    We present a statistical model designed to identify the effect of experimental perturbations on the aggregate behavior of the transcriptome expressed by the bacterium Dehalococcoides mccartyi strain 195. Strains of Dehalococcoides are used in sub-surface bioremediation applications because they organohalorespire tetrachloroethene and trichloroethene (common chlorinated solvents that contaminate the environment) to non-toxic ethene. However, the biochemical mechanism of this process remains incompletely described. Additionally, the response of Dehalococcoides to stress-inducing conditions that may be encountered at field-sites is not well understood. The constructed statistical model captured the aggregate behavior of gene expression phenotypes by modeling the distinct eigengenes of 100 transcript clusters, determining stable relationships among these clusters of gene transcripts with a sparse network-inference algorithm, and directly modeling the effect of changes in experimental conditions by constructing networks conditioned on the experimental state. Based on the model predictions, we discovered new response mechanisms for DMC, notably when the bacterium is exposed to solvent toxicity. The network identified a cluster containing thirteen gene transcripts directly connected to the solvent toxicity condition. Transcripts in this cluster include an iron-dependent regulator (DET0096-97) and a methylglyoxal synthase (DET0137). To validate these predictions, additional experiments were performed. Continuously fed cultures were exposed to saturating levels of tetrachloethene, thereby causing solvent toxicity, and transcripts that were predicted to be linked to solvent toxicity were monitored by quantitative reverse-transcription polymerase chain reaction. Twelve hours after being shocked with saturating levels of tetrachloroethene, the control transcripts (encoding for a key hydrogenase and the 16S rRNA) did not significantly change. By contrast, transcripts for DET0137 and DET0097 displayed a 46.8±11.5 and 14.6±9.3 fold up-regulation, respectively, supporting the model. This is the first study to identify transcripts in Dehalococcoides that potentially respond to tetrachloroethene solvent-toxicity conditions that may be encountered near contamination source zones in sub-surface environments.

  12. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    SciTech Connect (OSTI)

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  13. Summary Document: Restoration Plan for Major Airports after a Bioterrorist Attack

    SciTech Connect (OSTI)

    Raber, E

    2007-01-11

    This document provides general guidelines for developing a Restoration Plan for a major airport following release of a biological warfare agent. San Francisco International Airport was selected as the example airport during development of the Plan to illustrate specific details. The spore forming bacterium Bacillus anthracis was selected as the biological agent of primary concern because it is the most difficult of known bioterrorism agents to inactivate and is considered to be one of the agents most likely to be used as a biological weapon. The focus of the Plan is on activities associated with the Characterization, Remediation, and Clearance Phases that are defined herein. Activities associated with the Notification and First-Response Phases are briefly discussed in Appendixes A and B, respectively. In addition to the main text of this Plan and associated appendixes, a data supplement was developed specifically for San Francisco International Airport. Requests for the data supplement must be made directly to the Emergency Planning Operations Division of San Francisco International Airport.

  14. Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

    SciTech Connect (OSTI)

    Oosterkamp, Margreet J.; Veuskens, Teun; Saia, Flavia Talarico; Weelink, Sander A.B.; Goodwin, Lynne A.; Daligault, Hajnalka E.; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Land, Miriam L; Hauser, Loren John; Langenhoff, A. M.; Gerritse, Jan; Van Berkel, Willem J. H.; Pieper, Dietmar; Junca, Howard; Smidt, Hauke; Schraa, Gosse; Davids, Mark; Schaap, Peter J; Plugge, Caroline M.; Stams, Alfons J. M.

    2013-01-01

    The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.

  15. Cerebrospinal Fluid Proteome of Patients with Acute Lyme Disease

    SciTech Connect (OSTI)

    Angel, Thomas E.; Jacobs, Jon M.; Smith, Robert P.; Pasternack, Mark S.; Elias, Susan; Gritsenko, Marina A.; Shukla, Anil K.; Gilmore, Edward C.; McCarthy, Carol; Camp, David G.; Smith, Richard D.

    2012-10-05

    Acute Lyme disease results from transmission of and infection by the bacterium Borrelia burgdorferi following a tick bite. During acute infection, bacteria can disseminate to the central nervous system (CNS) leading to the development of Lyme meningitis. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing for a deep view into the proteome for a cohort of patients with early-disseminated Lyme disease and CSF inflammation leading to the identification of proteins that reflect host responses, which are distinct for subjects with acute Lyme disease. Additionally, we analyzed individual patient samples and quantified changes in protein abundance employing label-free quantitative mass spectrometry based methods. The measured changes in protein abundances reflect the impact of acute Lyme disease on the CNS as presented in CSF. We have identified 89 proteins that differ significantly in abundance in patients with acute Lyme disease. A number of the differentially abundant proteins have been found to be localized to brain synapse and thus constitute important leads for better understanding of the neurological consequence of disseminated Lyme disease.

  16. Cloning, expression, crystallization and preliminary X-ray analysis of a putative multiple antibiotic resistance repressor protein (MarR) from Xanthomonas campestris

    SciTech Connect (OSTI)

    Tu, Zhi-Le; Li, Juo-Ning; Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Lyu, Ping-Chiang; Gao, Fei Philip; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-07-01

    A putative repressor for the multiple antibiotic resistance operon from a plant pathogen X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. The crystals diffracted to 2.3 with good quality. The multiple antibiotic resistance operon (marRAB) is a member of the multidrug-resistance system. When induced, this operon enhances resistance of bacteria to a variety of medically important antibiotics, causing a serious global health problem. MarR is a marR-encoded protein that represses the transcription of the marRAB operon. Through binding with salicylate and certain antibiotics, however, MarR can derepress and activate the marRAB operon. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC1739, a putative MarR repressor protein present in the Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, are described. The XC1739 crystals diffracted to a resolution of at least 1.8 . They are orthorhombic and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 39.5, b = 54.2 and c = 139.5 , respectively. They contain two molecules in the asymmetric unit from calculation of the self-rotation function.

  17. Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola

    SciTech Connect (OSTI)

    O`Brien, D.A.

    1992-11-01

    A putative transcription factor in Rhodobactor capsulatus which binds upstream of the crt and bch pigment biosynthesis operons and appears to play a role in the adaptation of the organism from the aerobic to the anaerobic-photosynthetic growth mode was characterized. Chapter 2 describes the identification of this factor through an in vitro mobility shift assay, as well as the determination of its binding properties and sequence specificity. Chapter 3 focuses on the isolation of this factor. Biochemistry of later carotenoid biosynthesis enzymes derived from the non-photosynthetic bacterium, Erwinia herbicola. Chapter 4 describes the separate overexpression and in vitro analysis of two enzymes involved in the main sequence of the carotenoid biosynthesis pathway, lycopene cyclase and 5-carotene hydroxylase. Chapter 5 examines the overexpression and enzymology of functionally active zeaxanthin glucosyltransferase, an enzyme which carries out a more unusual transformation, converting a carotenoid into its more hydrophilic mono- and diglucoside derivatives. In addition, amino acid homology with other glucosyltransferases suggests a putative binding site for the UDP-activated glucose substrate.

  18. A Carotenoid-Deficient Mutant in Pantoea sp. YR343, a Bacteria Isolated from the Rhizosphere of Populus deltoides, Is Defective in Root Colonization

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Bible, Amber; Fletcher, Sarah J; Pelletier, Dale A; Schadt, Christopher Warren; Jawdy, Sara; Weston, David; Engle, Nancy L.; Tschaplinski, Timothy J.; Masyuko, Rachel; Polisetti, Sneha; et al

    2016-04-18

    The complex interactions between plants and their microbiome can have a profound effect on the health and productivity of the plant host. A better understanding of the microbial mechanisms that promote plant health and stress tolerance will enable strategies for improving the productivity of economically-important plants. Pantoea sp. YR343 is a motile, rod-shaped bacterium isolated from the roots of Populus deltoides that possesses the ability to solubilize phosphate and produce the phytohormone indole-3-acetic acid. Pantoea sp. YR343 readily colonizes plant roots and does not appear to be pathogenic when applied to the leaves or roots of selected plant hosts. Tomore » better understand the molecular mechanisms involved in plant association and rhizosphere survival by Pantoea sp. YR343, we constructed a mutant in which the crtB gene encoding phytoene synthase was deleted. Phytoene synthase is responsible for converting geranylgeranyl pyrophosphate to phytoene, an important precursor to the production of carotenoids. As predicted, the ΔcrtB mutant is defective in carotenoid production, and shows increased sensitivity to oxidative stress. Moreover, we find that the ΔcrtB mutant is impaired in biofilm formation and production of indole-3-acetic acid. Finally we demonstrate that the ΔcrtB mutant shows reduced colonization of plant roots. Taken together, these data suggest that carotenoids are important for plant association and/or rhizosphere survival in Pantoea sp. YR343.« less

  19. Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    McCaig, William D.; Loving, Crystal L.; Hughes, Holly R.; Brockmeier, Susan L.; Charbit, Alain

    2016-03-01

    Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structuresmore » has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Lastly, vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.« less

  20. Bioadsorption of rare earth elements through cell surface display of lanthanide binding tags

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Park, Dan M.; Reed, David W.; Yung, Mimi C.; Eslamimanesh, Ali; Lencka, Malgorzata M.; Anderko, Andrzej; Fujita, Yoshiko; Riman, Richard E.; Navrotsky, Alexandra; Jiao, Yongqin

    2016-02-02

    In this study, with the increasing demand for rare earth elements (REEs) in many emerging clean energy technologies, there is an urgent need for the development of new approaches for efficient REE extraction and recovery. As a step toward this goal, we genetically engineered the aerobic bacterium Caulobacter crescentus for REE adsorption through high-density cell surface display of lanthanide binding tags (LBTs) on its S-layer. The LBT-displayed strains exhibited enhanced adsorption of REEs compared to cells lacking LBT, high specificity for REEs, and an adsorption preference for REEs with small atomic radii. Adsorbed Tb3+ could be effectively recovered using citrate,more » consistent with thermodynamic speciation calculations that predicted strong complexation of Tb3+ by citrate. No reduction in Tb3+ adsorption capacity was observed following citrate elution, enabling consecutive adsorption/desorption cycles. The LBT-displayed strain was effective for extracting REEs from the acid leachate of core samples collected at a prospective rare earth mine. Our collective results demonstrate a rapid, efficient, and reversible process for REE adsorption with potential industrial application for REE enrichment and separation.« less

  1. A network biology approach to denitrification in Pseudomonas aeruginosa

    SciTech Connect (OSTI)

    Arat, Seda; Bullerjahn, George S.; Laubenbacher, Reinhard

    2015-02-23

    Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. aeruginosa can perform (complete) denitrification, a respiratory process of dissimilatory nitrate reduction to nitrogen gas via nitrite (NO?), nitric oxide (NO) and nitrous oxide (N?O). This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P. aeruginosa. To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen (O?), nitrate (NO?), and phosphate (PO?) suggests that PO? concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. aeruginosa under various environmental conditions, and these predictions are either experimentally validated or supported by pertinent biological literature. One motivation for this study is to capture the effect of PO? on a denitrification metabolic network of P. aeruginosa in order to shed light on mechanisms for greenhouse gas N?O accumulation during seasonal oxygen depletion in aquatic environments such as Lake Erie (Laurentian Great Lakes, USA). Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide.

  2. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    SciTech Connect (OSTI)

    Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2014-07-01

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed lectin-like domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.

  3. Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide

    SciTech Connect (OSTI)

    Bradshaw, William J.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2015-02-19

    Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket. In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to hostpathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.

  4. Adhesion and formation of microbial biofilms in complex microfluidic devices

    SciTech Connect (OSTI)

    Kumar, Aloke [ORNL; Karig, David K [ORNL; Neethirajan, Suresh [University of Guelph; Suresh, Anil K [ORNL; Srijanto, Bernadeta R [ORNL; Mukherjee, Partha P [ORNL; Retterer, Scott T [ORNL; Doktycz, Mitchel John [ORNL

    2012-01-01

    Shewanella oneidensis is a metal reducing bacterium, which is of interest for bioremediation and clean energy applications. S. oneidensis biofilms play a critical role in several situations such as in microbial energy harvesting devices. Here, we use a microfluidic device to quantify the effects of hydrodynamics on the biofilm morphology of S. oneidensis. For different rates of fluid flow through a complex microfluidic device, we studied the spatiotemporal dynamics of biofilms, and we quantified several morphological features such as spatial distribution, cluster formation and surface coverage. We found that hydrodynamics resulted in significant differences in biofilm dynamics. The baffles in the device created regions of low and high flow in the same device. At higher flow rates, a nonuniform biofilm develops, due to unequal advection in different regions of the microchannel. However, at lower flow rates, a more uniform biofilm evolved. This depicts competition between adhesion events, growth and fluid advection. Atomic force microscopy (AFM) revealed that higher production of extra-cellular polymeric substances (EPS) occurred at higher flow velocities.

  5. Field application of a genetically engineered microorganism for polycyclic aromatic hydrocarbon bioremediation process monitoring and control

    SciTech Connect (OSTI)

    Sayler, G.S.; Cox, C.D.; Ripp, S.; Nivens, D.E.; Werner, C.; Ahn, Y.; Matrubutham, U.; Burlage, R.

    1998-11-01

    On October 30, 1996, the US Environmental Protection Agency (EPA) commenced the first test release of genetically engineered microorganisms (GEMs) for use in bioremediation. The specific objectives of the investigation were multifaceted and include (1) testing the hypothesis that a GEM can be successfully introduced and maintained in a bioremediation process, (2) testing the concept of using, at the field scale, reporter organisms for direct bioremediation process monitoring and control, and (3) acquiring data that can be used in risk assessment decision making and protocol development for future field release applications of GEMs. The genetically engineered strain under investigation is Pseudomonas fluorescens strain HK44 (King et al., 1990). The original P. fluorescens parent strain was isolated from polycyclic aromatic hydrocarbon (PAH) contaminated manufactured gas plant soil. Thus, this bacterium is able to biodegrade naphthalene (as well as other substituted naphthalenes and other PAHs) and is able to function as a living bioluminescent reporter for the presence of naphthalene contamination, its bioavailability, and the functional process of biodegradation. A unique component of this field investigation was the availability of an array of large subsurface soil lysimeters. This article describes the experience associated with the release of a genetically modified microorganism, the lysimeter facility and its associated instrumentation, as well as representative data collected during the first eighteen months of operation.

  6. Crystallization and preliminary X-ray crystallographic analysis of MbtI, a protein essential for siderophore biosynthesis in Mycobacterium tuberculosis

    SciTech Connect (OSTI)

    Harrison, Anthony J.; Ramsay, Rochelle J.; Baker, Edward N.; Lott, J. Shaun

    2005-01-01

    MbtI, the putative isochorismate synthase essential for siderophore biosynthesis in M. tuberculosis, has been crystallized. Diffraction data have been collected to 1.8 resolution. Mycobacterium tuberculosis, the causative agent of tuberculosis, depends on the secretion of salicylate-based siderophores called mycobactins for the acquisition of extracellular iron, which is essential for the growth and virulence of the bacterium. The protein MbtI is thought to be the isochorismate synthase enzyme responsible for the conversion of chorismate to isochorismate, the first step in the salicylate production required for mycobactin biosynthesis. MbtI has been overexpressed in Escherichia coli, purified and crystallized. The crystals diffract to a maximum resolution of 1.8 . They belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 51.8, b = 163.4, c = 194.9 , consistent with the presence of either two, three or four molecules in the asymmetric unit.

  7. Horizontal gene transfer and the evolution of transcriptionalregulation in Escherichia coli

    SciTech Connect (OSTI)

    Price, Morgan N.; Dehal, Paramvir S.; Arkin, Adam P.

    2007-12-20

    Background: Most bacterial genes were acquired by horizontalgene transfer from other bacteria instead of being inherited bycontinuous vertical descent from an ancient ancestor}. To understand howthe regulation of these {acquired} genes evolved, we examined theevolutionary histories of transcription factors and of regulatoryinteractions from the model bacterium Escherichia coli K12. Results:Although most transcription factors have paralogs, these usually arose byhorizontal gene transfer rather than by duplication within the E. colilineage, as previously believed. In general, most neighbor regulators --regulators that are adjacent to genes that they regulate -- were acquiredby horizontal gene transfer, while most global regulators evolvedvertically within the gamma-Proteobacteria. Neighbor regulators wereoften acquired together with the adjacent operon that they regulate, sothe proximity might be maintained by repeated transfers (like "selfishoperons"). Many of the as-yet-uncharacterized (putative) regulators havealso been acquired together with adjacent genes, so we predict that theseare neighbor regulators as well. When we analyzed the histories ofregulatory interactions, we found that the evolution of regulation byduplication was rare, and surprisingly, many of the regulatoryinteractions that are shared between paralogs result from convergentevolution. Another surprise was that horizontally transferred genes aremore likely than other genes to be regulated by multiple regulators, andmost of this complex regulation probably evolved after the transfer.Conclusions: Our results highlight the rapid evolution of niche-specificgene regulation in bacteria.

  8. Complete genome sequence of Capnocytophaga ochracea type strain (VPI 2845T)

    SciTech Connect (OSTI)

    Mavromatis, K; Gronow, Sabine; Saunders, Elizabeth H; Land, Miriam L; Lapidus, Alla L.; Copeland, A; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Chen, Feng; Bruce, David; Tice, Hope; Cheng, Jan-Fang; Goodwin, Lynne A.; Pitluck, Sam; Pati, Amrita; Ivanova, N; Chen, Amy; Palaniappan, Krishna; Chain, Patrick S. G.; Hauser, Loren John; Chang, Yun-Juan; Jeffries, Cynthia; Brettin, Thomas S; Detter, J. Chris; Han, Cliff; Bristow, James; Goker, Markus; Eisen, Jonathan; Markowitz, Victor; Kyrpides, Nikos C; Klenk, Hans-Peter; Hugenholtz, Philip

    2009-01-01

    Capnocytophaga ochracea (Pr vot et al. 1956) Leadbetter et al. 1982 is the type species of the genus Capnocytophaga. It is of interest because of its location in the Flavobacteriaceae, a genomically not yet charted family within the order Flavobacteriales. The species grows as fusiform to rod shaped cells which tend to form clumps and are able to move by gliding. C. ochracea is known as a capnophilic (CO2-requiring) organism with the ability to grow under anaerobic as well as aerobic conditions (oxygen concentration larger than 15%), here only in the presence of 5% CO2. Strain VPI 2845T, the type strain of the species, is portrayed in this report as a gliding, Gram-negative bacterium, originally isolated from a human oral cavity. Here we describe the features of this organism, together with the complete genome se-quence, and annotation. This is the first completed genome sequence from the flavobacterial genus Capnocytophaga, and the 2,612,925 bp long single replicon genome with its 2193 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  9. Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome

    SciTech Connect (OSTI)

    Chhabra, S.R.; Butland, G.; Elias, D.; Chandonia, J.-M.; Fok, V.; Juba, T.; Gorur, A.; Allen, S.; Leung, C.-M.; Keller, K.; Reveco, S.; Zane, G.; Semkiw, E.; Prathapam, R.; Gold, B.; Singer, M.; Ouellet, M.; Sazakal, E.; Jorgens, D.; Price, M.; Witkowska, E.; Beller, H.; Hazen, T.C.; Biggin, M.; Auer, M.; Wall, J.; Keasling, J.

    2011-07-15

    The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

  10. Developing Research Capabilities in Energy Biosciences: Design principles of photosynthetic biofuel production.

    SciTech Connect (OSTI)

    Donald D. Brown; David Savage

    2012-06-30

    The current fossil fuel-based energy infrastructure is not sustainable. Solar radiation is a plausible alternative, but realizing it as such will require significant technological advances in the ability to harvest light energy and convert it into suitable fuels. The biological system of photosynthesis can carry out these reactions, and in principle could be engineered using the tools of synthetic biology. One desirable implementation would be to rewire the reactions of a photosynthetic bacterium to direct the energy harvested from solar radiation into the synthesis of the biofuel H2. Proposed here is a series of experiments to lay the basic science groundwork for such an attempt. The goal is to elucidate the transcriptional network of photosynthesis using a novel driver-reporter screen, evolve more robust hydrogenases for improved catalysis, and to test the ability of the photosynthetic machinery to directly produce H2 in vivo. The results of these experiments will have broad implications for the understanding of photosynthesis, enzyme function, and the engineering of biological systems for sustainable energy production. The ultimate impact could be a fundamental transformation of the world's energy economy.

  11. Changes in the composition of the human fecal microbiome following bacteriotherapy for recurrent Clostridium difficile-associated diarrhea

    SciTech Connect (OSTI)

    Khoruts, A.; Dicksved, J.; Jansson, J.K.; Sadowsky, M.J.

    2009-08-15

    CDAD is the major known cause of antibiotic-induced diarrhea and colitis, and the disease is thought to result from persistent disruption of commensal gut microbiota. Bacteriotherapy by way of fecal transplantation can be used to treat recurrent CDAD and is thought to re-establish the normal colonic microflora. However, limitations of conventional microbiologic techniques have until recently precluded testing of this idea. In this study we used T-RFLP and 16S rRNA gene sequencing approaches to characterize the bacterial composition of the colonic microflora in a patient suffering from recurrent CDAD, before and after treatment by fecal transplantation from a healthy donor. While the patient's residual colonic microbiota, prior to therapy, was deficient in members of the bacterial divisions-Firmicutes and Bacteriodetes, transplantation had a dramatic impact on the composition of the patient's gut microbiota. By 14 days post transplantation, the fecal bacterial composition of the recipient was highly similar to the donor and was dominated by Bacteroides spp. strains and an uncharacterized butyrate producing bacterium. The change in bacterial composition was accompanied by resolution of the patient's symptoms. The striking similarity of the recipient's and donor's intestinal microbiota following bacteriotherapy suggests that the donor's bacteria quickly occupied their requisite niches, resulting in restoration of both the structure and function of the microbial communities present.

  12. Spore Coat Architecture of Clostridium novyi-NT spores

    SciTech Connect (OSTI)

    Plomp, M; McCafferey, J; Cheong, I; Huang, X; Bettegowda, C; Kinzler, K; Zhou, S; Vogelstein, B; Malkin, A

    2007-05-07

    Spores of the anaerobic bacterium Clostridium novyi-NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Towards this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of dormant as well as germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers as well as the underlying spore coat and undercoat layers sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi-NT, these studies document the presence of proteinaceous growth spirals in a biological organism.

  13. Ecological succession and viability of human-associated microbiota on restroom surfaces

    SciTech Connect (OSTI)

    Gibbons, Sean M.; Schwartz, Tara; Fouquier, Jennifer; Mitchell, Michelle; Sangwan, Naseer; Gilbert, Jack A.; Kelley, Scott T.; Elkins, C. A.

    2014-11-14

    Human-associated bacteria dominate the built environment (BE). Following decontamination of floors, toilet seats, and soap dispensers in four public restrooms, in situ bacterial communities were characterized hourly, daily, and weekly to determine their successional ecology. The viability of cultivable bacteria, following the removal of dispersal agents (humans), was also assessed hourly. A late-successional community developed within 5 to 8 h on restroom floors and showed remarkable stability over weeks to months. Despite late-successional dominance by skin- and outdoor-associated bacteria, the most ubiquitous organisms were predominantly gut-associated taxa, which persisted following exclusion of humans. Staphylococcus represented the majority of the cultivable community, even after several hours of human exclusion. Methicillin-resistant Staphylococcus aureus (MRSA)-associated virulence genes were found on floors but were not present in assembled Staphylococcus pan-genomes. Viral abundances, which were predominantly enterophages, human papilloma virus, and herpesviruses, were significantly correlated with bacterial abundances and showed an unexpectedly low virus-to-bacterium ratio in surface-associated samples, suggesting that bacterial hosts are mostly dormant on BE surfaces.

  14. Cell fate regulation governed by a repurposed bacterial histidine kinase

    SciTech Connect (OSTI)

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  15. Analysis of a Ferric Uptake Regulator (Fur) Mutant ofDesulfovibrio vulgaris Hildenborough

    SciTech Connect (OSTI)

    Bender, Kelly S.; Yen, Huei-Che Bill; Hemme, Christopher L.; Yang, Zamin K.; He, Zhili; He, Qiang; Zhou, Jizhong; Huang, Katherine H.; Alm, Eric J.; Hazen, Terry C.; Arkin, Adam P.; Wall, Judy D.

    2007-09-21

    Previous experiments examining the transcriptional profileof the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of theFur regulon in response to various environmental stressors. To test theinvolvement of Fur in the growth response and transcriptional regulationof D. vulgaris, a targeted mutagenesis procedure was used for deletingthe fur gene. Growth of the resulting ?fur mutant (JW707) was notaffected by iron availability, but the mutant did exhibit increasedsensitivity to nitrite and osmotic stresses compared to the wild type.Transcriptional profiling of JW707 indicated that iron-bound Fur acts asa traditional repressor for ferrous iron uptake genes (feoAB) and othergenes containing a predicted Fur binding site within their promoter.Despite the apparent lack of siderophore biosynthesis genes within the D.vulgaris genome, a large 12-gene operon encoding orthologs to TonB andTolQR also appeared to be repressed by iron-bound Fur. While other genespredicted to be involved in iron homeostasis were unaffected by thepresence or absence of Fur, alternative expression patterns that could beinterpreted as repression or activation by iron-free Fur were observed.Both the physiological and transcriptional data implicate a globalregulatory role for Fur in the sulfate-reducing bacterium D.vulgaris.

  16. Analysis of Shewanella oneidensis Membrane Protein Expression in Response to Electron Acceptor Availability

    SciTech Connect (OSTI)

    Giometti, Carol S.; Khare, Tripti; Verberkmoes, Nathan; O'Loughlin, Ed; Lindberg, Carl; Thompson, Melissa; Hettich, Robert

    2006-04-05

    Shewanella oneidensis MR-1, a gram negative metal-reducing bacterium, can utilize a large number of electron acceptors. In the natural environment, S. oneidensis utilizes insoluble metal oxides as well as soluble terminal electron acceptors. The purpose of this ERSP project is to identify differentially expressed proteins associated with the membranes of S. oneidensis MR-1 cells grown with different electron acceptors, including insoluble metal oxides. We hypothesize that through the use of surface labeling, subcellular fractionation, and a combination of proteome analysis tools, proteins involved in the reduction of different terminal electron acceptors will be elucidated. We are comparing the protein profiles from cells grown with the soluble electron acceptors oxygen and fumarate and with those from cells grown with the insoluble iron oxides goethite, ferrihydrite and lepidocrocite. Comparison of the cell surface proteins isolated from cells grown with oxygen or anaerobically with fumarate revealed an increase in the abundance of over 25 proteins in anaerobic cells, including agglutination protein and flagellin proteins along with the several hypothetical proteins. In addition, the surface protein composition of cells grown with the insoluble iron oxides varies considerably from the protein composition observed with either soluble electron acceptor as well as between the different insoluble acceptors.

  17. Toward a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough

    SciTech Connect (OSTI)

    Chhabra, S.R.; Joachimiak, M.P.; Petzold, C.J.; Zane, G.M.; Price, M.N.; Gaucher, S.; Reveco, S.A.; Fok, V.; Johanson, A.R.; Batth, T.S.; Singer, M.; Chandonia, J.M.; Joyner, D.; Hazen, T.C.; Arkin, A.P.; Wall, J.D.; Singh, A.K.; Keasling, J.D.

    2011-05-01

    Proteinprotein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study E. coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio 5 vulgaris Hildenborough, a model anaerobe and sulfate reducer. In this paper we present the first attempt to identify protein-protein interactions in an obligate anaerobic bacterium. We used suicide vector-assisted chromosomal modification of 12 open reading frames encoded by this sulfate reducer to append an eight amino acid affinity tag to the carboxy-terminus of the chosen proteins. Three biological replicates of the 10 pulled-down proteins were separated and analyzed using liquid chromatography-mass spectrometry. Replicate agreement ranged between 35% and 69%. An interaction network among 12 bait and 90 prey proteins was reconstructed based on 134 bait-prey interactions computationally identified to be of high confidence. We discuss the biological significance of several unique metabolic features of D. vulgaris revealed by this protein-protein interaction data 15 and protein modifications that were observed. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.

  18. Soft rot decay capabilities and interactions of fungi and bacteria from fumigated utility poles

    SciTech Connect (OSTI)

    Wang, C.J.K.; Worrall, J.J. . Coll. of Environmental Science and Forestry)

    1992-11-01

    The objectives were to (1) identify microfungi and bacterial associates isolated from fumigated southern pine poles from EPRI project RP 1471-72, (2) study the soft-rot capabilities of predominant fungi, and (3) study interactions among microorganisms in relation to wood decay. Methods for identification followed standard techniques using morphological and physiological criteria. Soft-rot by microfungi alone and with bacteria was determined as weight loss and anatomical examination of wood blocks using light microscopy and limited electron microscopy. Acinetobacter calcoaceticus was the predominant bacterium. Twenty-one species of microfungi were identified including four new species. A book entitled IDENTIFICATION MANUAL FOR FUNGI FROM UTILITY POLES IN THE EASTERN UNITED STATES was published. An improved soft-rot test was devised. Fifty-one of 84 species (60%) of microfungi from poles tested were soft-rot positive; that is much greater than previously reported. Three types of anatomical damage of wood of pine or birch caused by soft-rot fungi were described. Interaction tests showed that, in some cases, there was a strong synergism between bacteria and fungi in causing weight loss, but results were inconsistent. Although soft rot is often most apparent under conditions of very high moisture, intermediate moisture levels appear to be optimal, as with basidiomycete decayers.

  19. Soft rot decay capabilities and interactions of fungi and bacteria from fumigated utility poles. Final report

    SciTech Connect (OSTI)

    Wang, C.J.K.; Worrall, J.J.

    1992-11-01

    The objectives were to (1) identify microfungi and bacterial associates isolated from fumigated southern pine poles from EPRI project RP 1471-72, (2) study the soft-rot capabilities of predominant fungi, and (3) study interactions among microorganisms in relation to wood decay. Methods for identification followed standard techniques using morphological and physiological criteria. Soft-rot by microfungi alone and with bacteria was determined as weight loss and anatomical examination of wood blocks using light microscopy and limited electron microscopy. Acinetobacter calcoaceticus was the predominant bacterium. Twenty-one species of microfungi were identified including four new species. A book entitled IDENTIFICATION MANUAL FOR FUNGI FROM UTILITY POLES IN THE EASTERN UNITED STATES was published. An improved soft-rot test was devised. Fifty-one of 84 species (60%) of microfungi from poles tested were soft-rot positive; that is much greater than previously reported. Three types of anatomical damage of wood of pine or birch caused by soft-rot fungi were described. Interaction tests showed that, in some cases, there was a strong synergism between bacteria and fungi in causing weight loss, but results were inconsistent. Although soft rot is often most apparent under conditions of very high moisture, intermediate moisture levels appear to be optimal, as with basidiomycete decayers.

  20. Whole-genome shotgun optical mapping of Rhodospirillum rubrum

    SciTech Connect (OSTI)

    Reslewic, S.; Zhou, S.; Place, M.; Zhang, Y.; Briska, A.; Goldstein, S.; Churas, C.; Runnheim, R.; Forrest, D.; Lim, A.; Lapidus, A.; Han, C. S.; Roberts, G. P.; Schwartz, D. C.

    2005-09-01

    Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.

  1. Spatial distribution of an uranium-respiring betaproteobacterium at the Rifle, CO field research site

    SciTech Connect (OSTI)

    Koribanics, Nicole M.; Tuorto, Steven J.; Lopez-Chiaffarelli, Nora; McGuinness, Lora R.; Häggblom, Max M.; Williams, Kenneth H.; Long, Philip E.; Kerkhof, Lee J.; Morais, Paula V

    2015-04-13

    The Department of Energy’s Integrated Field-Scale Subsurface Research Challenge Site (IFRC) at Rifle, Colorado was created to address the gaps in knowledge on the mechanisms and rates of U(VI) bioreduction in alluvial sediments. Previous studies at the Rifle IFRC have linked microbial processes to uranium immobilization during acetate amendment. Several key bacteria believed to be involved in radionuclide containment have been described; however, most of the evidence implicating uranium reduction with specific microbiota has been indirect. Here, we report on the cultivation of a microorganism from the Rifle IFRC that reduces uranium and appears to utilize it as a terminal electron acceptor for respiration with acetate as electron donor. Furthermore, this bacterium constitutes a significant proportion of the subsurface sediment community prior to biostimulation based on TRFLP profiling of 16S rRNA genes. 16S rRNA gene sequence analysis indicates that the microorganism is a betaproteobacterium with a high similarity to Burkholderia fungorum. This is, to our knowledge, the first report of a betaproteobacterium capable of uranium respiration. Our results indicate that this microorganism occurs commonly in alluvial sediments located between 3-6 m below ground surface at Rifle and may play a role in the initial reduction of uranium at the site.

  2. Complete genome sequence of Capnocytophaga ochracea type strain (VPI 2845T)

    SciTech Connect (OSTI)

    Mavromatis, Konstantinos; Gronow, Sabine; Saunders, Elizabeth; Land, Miriam; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Chen, Feng; Tice1, Hope; Cheng, Jan-Fang; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Pati, Amrita; Ivanova, Natalia; Chen, Amy; Palaniappan, Krishna; Chain, Patrick; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C.; Brettin, Thomas; Detter, John C.; Han, Cliff; Bristow, James; Goker, Markus; Rohde, Manfred; Eisen, Jonathan A.; Markowitz, Victor; Kyrpides, Nikos C.; Klenk, Hans-Peter; Hugenholtz, Philip

    2009-05-20

    Capnocytophaga ochracea (Prevot et al. 1956) Leadbetter et al. 1982 is the type species of the genus Capnocytophaga. It is of interest because of its location in the Flavobacteriaceae, a genomically yet uncharted family within the order Flavobacteriales. The species grows as fusiform to rod shaped cells which tend to form clumps and are able to move by gliding. C. ochracea is known as a capnophilic organism with the ability to grow under anaerobic as well as under aerobic conditions (oxygen concentration larger than 15percent), here only in the presence of 5percent CO2. Strain VPI 2845T, the type strain of the species, is portrayed in this report as a gliding, Gram-negative bacterium, originally isolated from a human oral cavity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the flavobacterial genus Capnocytophaga, and the 2,612,925 bp long single replicon genome with its 2193 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  3. Complete genome sequence of Actinosynnema mirum type strain (101T)

    SciTech Connect (OSTI)

    Land, Miriam; Lapidus, Alla; Mayilraj, Shanmugam; Chen, Feng; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Chertkov, Olga; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Rohde, Manfred; Goker, Markus; Pati, Amrita; Ivanova, Natalia; Mavrommatis, Konstantinos; Chen, Amy; Palaniappan, Krishna; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia; Brettin, Thomas; Detter, John C.; Han, Cliff; Chain, Patrick; Tindall, Brian; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter

    2009-05-20

    Actinosynnema mirum Hasegawa et al. 1978 is the type species of the genus, and is of phylogenetic interest because of its central phylogenetic location in the Actino-synnemataceae, a rapidly growing family within the actinobacterial suborder Pseudo-nocardineae. A. mirum is characterized by its motile spores borne on synnemata and as a producer of nocardicin antibiotics. It is capable of growing aerobically and under a moderate CO2 atmosphere. The strain is a Gram-positive, aerial and substrate mycelium producing bacterium, originally isolated from a grass blade collected from the Raritan River, New Jersey. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Actinosynnemataceae, and only the second sequence from the actinobacterial suborder Pseudonocardineae. The 8,248,144 bp long single replicon genome with its 7100 protein-coding and 77 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  4. Complete genome sequence of Kytococcus sedentarius type strain (strain 541T)

    SciTech Connect (OSTI)

    Sims, David; Brettin, Thomas; Detter, John C.; Han, Cliff; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Chen, Feng; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ovchinnikova, Galina; Pati, Amrita; Ivanova, Natalia; Mavrommatis, Konstantinos; Chen, Amy; Palaniappan, Krishna; D'haeseleer, Patrick; Chain, Patrick; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Schneider, Susanne; Goker, Markus; Pukall, Rudiger; Kyrpides, Nikos C.; Klenk, Hans-Peter

    2009-05-20

    Kytococcus sedentarius (ZoBell and Upham 1944) Stackebrandt et al. 1995 is the type strain of the species, and is of phylogenetic interest because of its location in the Dermacoccaceae, a poorly studied family within the actinobacterial suborder Micrococcineae. K. sedentarius is known for the production of oligoketide antibiotics as well as for its role as an opportunistic pathogen causing valve endocarditis, hemorrhagic pneumonia, and pitted keratolysis. It is strictly aerobic and can only grow when several amino acids are provided in the medium. The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, originally isolated from a marine environment. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Dermacoccaceae and the 2,785,024 bp long single replicon genome with its 2639 protein-coding and 64 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  5. Complete genome sequence of Desulfomicrobium baculatum type strain (XT)

    SciTech Connect (OSTI)

    Copeland, Alex; Spring, Stefan; Goker, Markus; Schneider, Susanne; Lapidus, Alla; Glavina Del Rio, Tijana; Tice, Hope; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavrommatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C; Meincke, Linda; Sims, David; Brettin, Thomas; Detter, John C; Han, Cliff; Chain, Patrick; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C; Lucas, Susan

    2009-05-20

    Desulfomicrobium baculatum is the type species of the genus Desulfomicrobium, which is the type genus of the family Desulfomicrobiaceae. It is of phylogenetic interest because of the isolated location of the family Desulfomicrobiaceae within the order Desulfovibrionales. D. baculatum strain XT is a Gram-negative, motile, sulfate-reducing bacterium isolated from water-saturated manganese carbonate ore. It is strictly anaerobic and does not require NaCl for growth, although NaCl concentrations up to 6percent (w/v) are tolerated. The metabolism is respiratory or fermentative. In the presence of sulfate, pyruvate and lactate are incompletely oxidized to acetate and CO2. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the deltaproteobacterial family Desulfomicrobiaceae, and this 3,942,657 bp long single replicon genome with its 3494 protein-coding and 72 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  6. Novel methods for detecting buried explosive devices

    SciTech Connect (OSTI)

    Kercel, S.W.; Burlage, R.S.; Patek, D.R.; Smith, C.M.; Hibbs, A.D.; Rayner, T.J.

    1997-04-01

    Oak Ridge National Laboratory (ORNL) and Quantum Magnetics, Inc. (QM) are exploring novel landmine detection technologies. Technologies considered here include bioreporter bacteria, swept acoustic resonance, nuclear quadrupole resonance (NQR), and semiotic data fusion. Bioreporter bacteria look promising for third-world humanitarian applications; they are inexpensive, and deployment does not require high-tech methods. Swept acoustic resonance may be a useful adjunct to magnetometers in humanitarian demining. For military demining, NQR is a promising method for detecting explosive substances; of 50,000 substances that have been tested, none has an NQR signature that can be mistaken for RDX or TNT. For both military and commercial demining, sensor fusion entails two daunting tasks, identifying fusible features in both present-day and emerging technologies, and devising a fusion algorithm that runs in real-time on cheap hardware. Preliminary research in these areas is encouraging. A bioreporter bacterium for TNT detection is under development. Investigation has just started in swept acoustic resonance as an approach to a cheap mine detector for humanitarian use. Real-time wavelet processing appears to be a key to extending NQR bomb detection into mine detection, including TNT-based mines. Recent discoveries in semiotics may be the breakthrough that will lead to a robust fused detection scheme.

  7. Structure-toxicity assessment of metabolites of the aerobic bacterial transformation of substituted naphthalenes

    SciTech Connect (OSTI)

    LeBlond, J.D.; Applegate, B.M.; Menn, F.M.; Schultz, T.W.; Sayler, G.S.

    2000-05-01

    Pseudomonas fluorescens 5R, a naphthalene-degrading bacterium isolated from manufactured gas plant soil contaminated with polycyclic aromatic hydrocarbons, was examined for its degradative capacity of a number of substituted naphthalenes. In general, those compounds substituted on only one ring with an electrically neutral substituent were found to be transformed primarily to substituted salicylic acids according to the classical (NAH7) naphthalene dioxygenase-initiated upper pathway reactions of the naphthalene degradative pathway (i.e., the NAH system). Dimethylnaphthalenes with a substituent on each ring, and certain halogenated naphthalenes, were transformed via a monohydroxylation reaction to form hydroxylated dead-end products. Of the substituted salicylic acids examined, only 3- and 4-methylsalicylic acid, the respective products of the degradation of 1- and 2-methylnaphthalene, were further degraded by salicylate hydroxylase and catechol 2,3-dioxygenase, the first two enzymes of the NAH lower pathway. Using the Tetrahymena pyriformis acute toxicity assay, many of the monohydroxylated products of incomplete biodegradation were found to be polar narcotics. Substituted salicylic acids that are not further degraded by the NAH lower pathway were found to be toxic via carboxylic acid narcosis.

  8. Genome analysis of Elusimicrobium minutum, the first cultivated representative of the Elusimicrobia phylum (formerly Termite Group 1)

    SciTech Connect (OSTI)

    Herlemann, D. P. R.; Geissinger, O.; Ikeda-Ohtsubo, W.; Kunin, V.; Sun, H.; Lapidus, A.; Hugenholtz, P.; Brune, A.

    2009-02-01

    The candidate phylum Termite group 1 (TG1), is regularly 1 encountered in termite hindguts but is present also in many other habitats. Here we report the complete genome sequence (1.64 Mbp) of Elusimicrobium minutum strain Pei191{sup T}, the first cultured representative of the TG1 phylum. We reconstructed the metabolism of this strictly anaerobic bacterium isolated from a beetle larva gut and discuss the findings in light of physiological data. E. minutum has all genes required for uptake and fermentation of sugars via the Embden-Meyerhof pathway, including several hydrogenases, and an unusual peptide degradation pathway comprising transamination reactions and leading to the formation of alanine, which is excreted in substantial amounts. The presence of genes encoding lipopolysaccharide biosynthesis and the presence of a pathway for peptidoglycan formation are consistent with ultrastructural evidence of a Gram-negative cell envelope. Even though electron micrographs showed no cell appendages, the genome encodes many genes putatively involved in pilus assembly. We assigned some to a type II secretion system, but the function of 60 pilE-like genes remains unknown. Numerous genes with hypothetical functions, e.g., polyketide synthesis, non-ribosomal peptide synthesis, antibiotic transport, and oxygen stress protection, indicate the presence of hitherto undiscovered physiological traits. Comparative analysis of 22 concatenated single-copy marker genes corroborated the status of Elusimicrobia (formerly TG1) as a separate phylum in the bacterial domain, which was so far based only on 16S rRNA sequence analysis.

  9. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    SciTech Connect (OSTI)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  10. Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

    SciTech Connect (OSTI)

    Higgins, M.; Whitworth, G; El Warry, N; Randriantsoa, M; Samain, E; Burke, R; Vocadlo, D; Boraston, A

    2009-01-01

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  11. Book review of Insect Symbiosis. Volume 2. Bourtzis, K.A. and Miller, T.A. editros. 2006 CRC Press, Taylor and Francis Group, Boca Raton, FL, 276 pp. ISBN 0-8493-1286-8

    SciTech Connect (OSTI)

    Hoy, M.A. [Department of Entomology and Nematology, University of Florida, Gainesville, FL (United States)

    2007-03-15

    There are several definitions of symbiosis, but in this book it involves an association where one organism (the symbiont) lives within or on the body of another organism (the host), regardless of the actual effect on the host. Some symbioses are mutualistic, some parasitic, and some involve commensalism, in which one partner derives some benefit without either harming or benefiting the other. This is the second volume in this exciting and rapidly advancing topic by these editors. The first volume was published in 2003 and during the intervening three years additional data have been produced that make this book a useful addition to your library. The first book provided chapters that provided an overview of insect symbiosis, discussions of the primary aphid symbiont Buchnera and other aphid symbionts, symbiosis in tsetse, symbionts in the weevil Sitophilus , the possible use of paratransgenic symbionts of Rhodnius prolixis to prevent disease transmission, bark beetle and fungal symbiosis, symbionts of tephritid fruit flies, symbionts affecting termite behavior, an overview of microsporidia as symbionts (parasites?) of insects, an overview of a newly discovered bacterium that causes sex-ratio distortion in insects and mites (from the Bacteroides group), symbionts that selectively kill male insects, and several chapters on the ubiquitous endosymbiont Wolbachia.

  12. Cell fate regulation governed by a repurposed bacterial histidine kinase

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interactionmore » between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.« less

  13. Ecological succession and viability of human-associated microbiota on restroom surfaces

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Gibbons, Sean M.; Schwartz, Tara; Fouquier, Jennifer; Mitchell, Michelle; Sangwan, Naseer; Gilbert, Jack A.; Kelley, Scott T.; Elkins, C. A.

    2014-11-14

    Human-associated bacteria dominate the built environment (BE). Following decontamination of floors, toilet seats, and soap dispensers in four public restrooms, in situ bacterial communities were characterized hourly, daily, and weekly to determine their successional ecology. The viability of cultivable bacteria, following the removal of dispersal agents (humans), was also assessed hourly. A late-successional community developed within 5 to 8 h on restroom floors and showed remarkable stability over weeks to months. Despite late-successional dominance by skin- and outdoor-associated bacteria, the most ubiquitous organisms were predominantly gut-associated taxa, which persisted following exclusion of humans. Staphylococcus represented the majority of the cultivablemore » community, even after several hours of human exclusion. Methicillin-resistant Staphylococcus aureus (MRSA)-associated virulence genes were found on floors but were not present in assembled Staphylococcus pan-genomes. Viral abundances, which were predominantly enterophages, human papilloma virus, and herpesviruses, were significantly correlated with bacterial abundances and showed an unexpectedly low virus-to-bacterium ratio in surface-associated samples, suggesting that bacterial hosts are mostly dormant on BE surfaces.« less

  14. Alteration of Iron-Rich Lacustrine Sediments by Dissimilatory Iron-Reducing Bacteria

    SciTech Connect (OSTI)

    Crowe,S.; Roberts, J.; Weisener, C.; Fowle, D.

    2007-01-01

    The reduction of Fe during bacterial anaerobic respiration in sediments and soils not only causes the degradation of organic matter but also results in changes in mineralogy and the redistribution of many nutrients and trace metals. Understanding trace metal patterns in sedimentary rocks and predicting the fate of contaminants in the environment requires a detailed understanding of the mechanisms through which they are redistributed during Fe reduction. In this work, lacustrine sediments from Lake Matano in Indonesia were incubated in a minimal media with the dissimilatory iron reducing (DIR) bacterium Shewanella putrefaciens 200R. These sediments were reductively dissolved at rates slower than pure synthetic goethite despite the presence of an 'easily reducible' component, as defined by selective extractions. DIR of the lacustrine sediments resulted in the substrate-dependent production of abundant quantities of extracellular polymeric substances. Trace elements, including Ni, Co, P, Si, and As, were released from the sediments with progressive Fe reduction while Cr was sequestered. Much of the initial trace metal mobility can be attributed to the rapid reduction of a Mn-rich oxyhydroxide phase. The production of organo-Fe(III) reveals that DIR bacteria can generate significant metal complexation capacity. This work demonstrates that DIR induces the release of many elements associated with Fe-Mn oxyhydroxides, despite secondary mineralization.

  15. Spatial distribution of an uranium-respiring betaproteobacterium at the Rifle, CO field research site

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Koribanics, Nicole M.; Tuorto, Steven J.; Lopez-Chiaffarelli, Nora; McGuinness, Lora R.; Häggblom, Max M.; Williams, Kenneth H.; Long, Philip E.; Kerkhof, Lee J.; Morais, Paula V

    2015-04-13

    The Department of Energy’s Integrated Field-Scale Subsurface Research Challenge Site (IFRC) at Rifle, Colorado was created to address the gaps in knowledge on the mechanisms and rates of U(VI) bioreduction in alluvial sediments. Previous studies at the Rifle IFRC have linked microbial processes to uranium immobilization during acetate amendment. Several key bacteria believed to be involved in radionuclide containment have been described; however, most of the evidence implicating uranium reduction with specific microbiota has been indirect. Here, we report on the cultivation of a microorganism from the Rifle IFRC that reduces uranium and appears to utilize it as a terminalmore » electron acceptor for respiration with acetate as electron donor. Furthermore, this bacterium constitutes a significant proportion of the subsurface sediment community prior to biostimulation based on TRFLP profiling of 16S rRNA genes. 16S rRNA gene sequence analysis indicates that the microorganism is a betaproteobacterium with a high similarity to Burkholderia fungorum. This is, to our knowledge, the first report of a betaproteobacterium capable of uranium respiration. Our results indicate that this microorganism occurs commonly in alluvial sediments located between 3-6 m below ground surface at Rifle and may play a role in the initial reduction of uranium at the site.« less

  16. A network biology approach to denitrification in Pseudomonas aeruginosa

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Arat, Seda; Bullerjahn, George S.; Laubenbacher, Reinhard

    2015-02-23

    Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. aeruginosa can perform (complete) denitrification, a respiratory process of dissimilatory nitrate reduction to nitrogen gas via nitrite (NO₂), nitric oxide (NO) and nitrous oxide (N₂O). This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P. aeruginosa. To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen (O₂), nitrate (NO₃),more » and phosphate (PO₄) suggests that PO₄ concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. aeruginosa under various environmental conditions, and these predictions are either experimentally validated or supported by pertinent biological literature. One motivation for this study is to capture the effect of PO₄ on a denitrification metabolic network of P. aeruginosa in order to shed light on mechanisms for greenhouse gas N₂O accumulation during seasonal oxygen depletion in aquatic environments such as Lake Erie (Laurentian Great Lakes, USA). Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide.« less

  17. Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

    SciTech Connect (OSTI)

    Yung, M C; Jiao, Y

    2014-07-22

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria.

  18. Genome-scale reconstruction of the metabolic network in Yersinia pestis, strain 91001

    SciTech Connect (OSTI)

    Navid, A; Almaas, E

    2009-01-13

    The gram-negative bacterium Yersinia pestis, the aetiological agent of bubonic plague, is one the deadliest pathogens known to man. Despite its historical reputation, plague is a modern disease which annually afflicts thousands of people. Public safety considerations greatly limit clinical experimentation on this organism and thus development of theoretical tools to analyze the capabilities of this pathogen is of utmost importance. Here, we report the first genome-scale metabolic model of Yersinia pestis biovar Mediaevalis based both on its recently annotated genome, and physiological and biochemical data from literature. Our model demonstrates excellent agreement with Y. pestis known metabolic needs and capabilities. Since Y. pestis is a meiotrophic organism, we have developed CryptFind, a systematic approach to identify all candidate cryptic genes responsible for known and theoretical meiotrophic phenomena. In addition to uncovering every known cryptic gene for Y. pestis, our analysis of the rhamnose fermentation pathway suggests that betB is the responsible cryptic gene. Despite all of our medical advances, we still do not have a vaccine for bubonic plague. Recent discoveries of antibiotic resistant strains of Yersinia pestis coupled with the threat of plague being used as a bioterrorism weapon compel us to develop new tools for studying the physiology of this deadly pathogen. Using our theoretical model, we can study the cell's phenotypic behavior under different circumstances and identify metabolic weaknesses which may be harnessed for the development of therapeutics. Additionally, the automatic identification of cryptic genes expands the usage of genomic data for pharmaceutical purposes.

  19. Final Technical Report

    SciTech Connect (OSTI)

    David B. Wilson

    2008-04-02

    This grant provided the basic funding that enabled me to carry out a detailed characterization of the proteins used by the aerobic soil bacterium, Thermobifida fusca, to degrade cellulose and to study the mechanisms used by T. fusca to regulate cellulase synthesis. This work resulted in 53 publications and led to the decision by The DOE Joint Genome Institute to sequence the T. fusca genome. T. fusca is now recognized as one of the best studied cellulolytic microorganisms and our work led to the discovery of a novel class of cellulases, processive endoglucanases, which are found in many cellulolytic bacteria including both aerobes and anaerobes. In addition, we were able to determine the mechanism by which Cel9A caused processive hydrolysis of cellulose. This research also helped to explain why many cellulolytic microorganisms produce two different exocellulases, as we showed that these enzymes have different specificities, with one attacking the reducing end of a cellulose chain and the other attacking the nonreducing end. Our work also provided additional evidence for the importance of a cellulose binding domain (carbohydrate binding module) [CBM] in the hydrolysis of crystalline cellulose.

  20. Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

    SciTech Connect (OSTI)

    Li, Wang; College of Animal Science, Henan Science and Technology University, Luoyang 471003 ; Huan, Xiajuan; Zhou, Ying; Ma, Qingyi; Chen, Yulin

    2009-06-12

    A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.

  1. Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

    SciTech Connect (OSTI)

    Dykstra, Andrew B; Rodriguez, Jr., Miguel; Raman, Babu; Cook, Kelsey; Hettich, Robert {Bob} L

    2013-01-01

    Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to characterize the post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally-activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of multiple enzymes and fragmentation technologies allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

  2. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    SciTech Connect (OSTI)

    Guss, Adam M; Olson, Daniel G.; Caiazza, Nicky; Lynd, Lee R

    2012-01-01

    BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  3. Probing the mechanism of cellulosome attachment to the Clostridium thermocellum cell surface: computer simulation of the Type II Cohesin-Dockerin complex and its variants

    SciTech Connect (OSTI)

    Xu, Jiancong; Smith, Jeremy C

    2010-10-01

    The recalcitrance of lignocellulosic biomass to hydrolysis is the bottleneck in cellulosic ethanol production. Efficient degradation of biomass by the anaerobic bacterium Clostridium thermocellum is carried out by the multicomponent cellulosome complex. The bacterial cell-surface attachment of the cellulosome is mediated by high-affinity protein-protein interactions between the Type II cohesin domain borne by the cell envelope protein and the Type II dockerin domain, together with neighboring X-module present at the C-terminus of the scaffolding protein (Type II coh-Xdoc). Here, the Type II coh-Xdoc interaction is probed using molecular dynamics simulations, free-energy calculations and essential dynamics analyses on both the wild type and various mutants of the C. thermocellum Type II coh-Xdoc in aqueous solution. The simulations identify the hot spots, i.e. the amino acid residues that may lead to a dramatic decrease in binding affinity upon mutation and also probe the effects of mutations on the mode of binding. The results suggest that bulky and hydrophobic residues at the protein interface, which make specific contacts with their counterparts, may play essential roles in retaining a rigid cohesin-dockerin interface. Moreover, dynamical cross-correlation analysis indicates that the X-module has a dramatic effect on the cohesin-dockerin interaction and is required for the dynamical integrity of the interface.

  4. Biosynthesis of titanium dioxide nanoparticles using a probiotic from coal fly ash effluent

    SciTech Connect (OSTI)

    Babitha, S; Korrapati, Purna Sai

    2013-11-15

    Graphical abstract: - Highlights: Metal resistant probiotic species was isolated from coal fly ash effluent site. Uniform sized anatase form of TiO{sub 2} nanoparticles were synthesized using Propionibacterium jensenii. Diffraction patterns confirmed the anatase TiO{sub 2} NPs with average size <80 nm. TiO{sub 2} nanoparticle incorporated wound dressing exhibits better wound healing. - Abstract: The synthesis of titanium dioxide nanoparticle (TiO{sub 2} NP) has gained importance in the recent years owing to its wide range of potential biological applications. The present study demonstrates the synthesis of TiO{sub 2} NPs by a metal resistant bacterium isolated from the coal fly ash effluent. This bacterial strain was identified on the basis of morphology and 16s rDNA gene sequence [KC545833]. The physico-chemical characterization of the synthesized nanoparticles is completely elucidated by energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FTIR) and transmission and scanning electron microscopy (TEM, SEM). The crystalline nature of the nanoparticles was confirmed by X-RD pattern. Further, cell viability and haemolytic assays confirmed the biocompatible and non toxic nature of the NPs. The TiO{sub 2} NPs was found to enhance the collagen stabilization and thereby enabling the preparation of collagen based biological wound dressing. The paper essentially provides scope for an easy bioprocess for the synthesis of TiO{sub 2} NPs from the metal oxide enriched effluent sample for future biological applications.

  5. Nitrogen availability and indirect measurements of greenhouse gas emissions from aerobic and anaerobic biowaste digestates applied to agricultural soils

    SciTech Connect (OSTI)

    Rigby, H.; Smith, S.R.

    2013-12-15

    Highlights: Nitrogen release in digestate-amended soil depends on the digestate type. Overall N release is modulated by digestate mineral and mineralisable N contents. Microbial immobilisation does not influence overall release of digestate N in soil. Digestate physical properties and soil type interact to affect overall N recovery. High labile C inputs in digestate may promote denitrification in fine-textured soil. - Abstract: Recycling biowaste digestates on agricultural land diverts biodegradable waste from landfill disposal and represents a sustainable source of nutrients and organic matter (OM) to improve soil for crop production. However, the dynamics of nitrogen (N) release from these organic N sources must be determined to optimise their fertiliser value and management. This laboratory incubation experiment examined the effects of digestate type (aerobic and anaerobic), waste type (industrial, agricultural and municipal solid waste or sewage sludge) and soil type (sandy loam, sandy silt loam and silty clay) on N availability in digestate-amended soils and also quantified the extent and significance of the immobilisation of N within the soil microbial biomass, as a possible regulatory mechanism of N release. The digestate types examined included: dewatered, anaerobically digested biosolids (DMAD); dewatered, anaerobic mesophilic digestate from the organic fraction of municipal solid waste (DMADMSW); liquid, anaerobic co-digestate of food and animal slurry (LcoMAD) and liquid, thermophilic aerobic digestate of food waste (LTAD). Ammonium chloride (NH{sub 4}Cl) was included as a reference treatment for mineral N. After 48 days, the final, maximum net recoveries of mineral N relative to the total N (TN) addition in the different digestates and unamended control treatments were in the decreasing order: LcoMAD, 68%; LTAD, 37%, DMAD, 20%; and DMADMSW, 11%. A transient increase in microbial biomass N (MBN) was observed with LTAD application, indicating greater microbial activity in amended soil and reflecting the lower stability of this OM source, compared to the other, anaerobic digestate types, which showed no consistent effects on MBN compared to the control. Thus, the overall net release of digestate N in different soil types was not regulated by N transfer into the soil microbial biomass, but was determined primarily by digestate properties and the capacity of the soil type to process and turnover digestate N. In contrast to the sandy soil types, where nitrate (NO{sub 3}{sup -}) concentrations increased during incubation, there was an absence of NO{sub 3}{sup -} accumulation in the silty clay soil amended with LTAD and DMADMSW. This provided indirect evidence for denitrification activity and the gaseous loss of N, and the associated increased risk of greenhouse gas emissions under certain conditions of labile C supply and/or digestate physical structure in fine-textured soil types. The significance and influence of the interaction between soil type and digestate stability and physical properties on denitrification processes in digestate-amended soils require urgent investigation to ensure management practices are appropriate to minimise greenhouse gas emissions from land applied biowastes.

  6. Integrated genome-based studies of Shewanella ecophysiology

    SciTech Connect (OSTI)

    Segre Daniel; Beg Qasim

    2012-02-14

    This project was a component of the Shewanella Federation and, as such, contributed to the overall goal of applying the genomic tools to better understand eco-physiology and speciation of respiratory-versatile members of Shewanella genus. Our role at Boston University was to perform bioreactor and high throughput gene expression microarrays, and combine dynamic flux balance modeling with experimentally obtained transcriptional and gene expression datasets from different growth conditions. In the first part of project, we designed the S. oneidensis microarray probes for Affymetrix Inc. (based in California), then we identified the pathways of carbon utilization in the metal-reducing marine bacterium Shewanella oneidensis MR-1, using our newly designed high-density oligonucleotide Affymetrix microarray on Shewanella cells grown with various carbon sources. Next, using a combination of experimental and computational approaches, we built algorithm and methods to integrate the transcriptional and metabolic regulatory networks of S. oneidensis. Specifically, we combined mRNA microarray and metabolite measurements with statistical inference and dynamic flux balance analysis (dFBA) to study the transcriptional response of S. oneidensis MR-1 as it passes through exponential, stationary, and transition phases. By measuring time-dependent mRNA expression levels during batch growth of S. oneidensis MR-1 under two radically different nutrient compositions (minimal lactate and nutritionally rich LB medium), we obtain detailed snapshots of the regulatory strategies used by this bacterium to cope with gradually changing nutrient availability. In addition to traditional clustering, which provides a first indication of major regulatory trends and transcription factors activities, we developed and implemented a new computational approach for Dynamic Detection of Transcriptional Triggers (D2T2). This new method allows us to infer a putative topology of transcriptional dependencies, with special emphasis on the nodes at which external stimuli are expected to affect the internal dynamics. In parallel, we addressed the question of how to compare transcriptional profiles across different time-course experiments. Our growth derivative mapping (GDM) method makes it possible to relate with each other points that correspond to the same relative growth rate in different media sets. This mapping allowed us to discriminate between genes that display an environment-independent behavior, and genes whose transcription seems to be tuned by specific environmental factors. Our analysis highlighted the importance of some specific pathways, whose metabolic relevance was confirmed by dynamic flux balance analysis (dFBA) calculations. In particular, we found that oxygen limitation potentially triggers the activation of genes previously shown to be relevant for anaerobic respiration, and that nitrogen limitation is coupled to storage of glycogen. Both observations have been corroborated by measurement of relevant intracellular and extracellular metabolites, as well as by complementary analyses of literature information and competitive fitness assay data. The pipeline of experimental and computational approaches applied and developed for this work could be extended to other microbes and additional conditions.

  7. Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; Tomchick, Diana R.; Norgard, Michael V.

    2015-05-05

    The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

  8. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

    SciTech Connect (OSTI)

    Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; Martí-Arbona, Ricardo

    2015-09-18

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

  9. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway

    SciTech Connect (OSTI)

    Webb, N.A.; Mulichak, A.M.; Lam, J.S.; Rocchetta, H.L.; Garavito, R.M.

    2010-03-08

    D-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthesis of D-rhamnose proceeds through the conversion of GDP-D-mannose by GDP-D-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-D-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP. GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr). Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana. The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 {angstrom} of each other. A short peptide segment (Arg35-Arg43) stretches into the neighboring monomer, making not only protein-protein interactions but also hydrogen bonding interactions with the neighboring cofactor. The interface hydrogen bonds made by the Arg35-Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs. Outside of the Arg35-Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species. These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.

  10. Genomic Sequencing of Single Microbial Cells from Environmental Samples

    SciTech Connect (OSTI)

    Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

    2008-02-01

    Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

  11. The Effect of Bicarbonate on the Microbial Dissolution of Autunite Mineral in the Presence of Gram-Positive Bacteria

    SciTech Connect (OSTI)

    Sepulveda-Medina, Paola; Katsenovich, Yelena; Wellman, Dawn M.; Lagos, Leonel

    2015-06-01

    Bacteria are key players in the processes that govern fate and transport of contaminants. The uranium release from Na and Ca-autunite by Arthrobacter oxydans strain G968 was evaluated in the presence of bicarbonate ions. This bacterium was previously isolated from Hanford Site soil and in earlier prescreening tests demonstrated low tolerance to U(VI) toxicity compared to other A.oxydans isolates. Experiments were conducted using glass serum bottles as mixed bioreactors and sterile 6-well cell culture plates with inserts separating bacteria cells from mineral solids. Reactors containing phosphorus-limiting media were amended with bicarbonate ranging between 0-10 mM and metaautunite solids to provide a U(VI) concentration of 4.4 mmol/L. Results showed that in the presence of bicarbonate, A.oxydans G968 was able to enhance the release of U(VI) from Na and Ca autunite at the same capacity as other A.oxydans isolates with relatively high tolerance to U(VI). The effect of bacterial strains on autunite dissolution decreases as the concentration of bicarbonate increases. The results illustrate that direct interaction between the bacteria and the mineral is not necessary to result in U (VI) biorelease from autunite. The formation of secondary calcium-phosphate mineral phases on the surface of the mineral during the dissolution can ultimately reduce the natural autunite mineral contact area, which bacterial cells can access. This thereby reduces the concentration of uranium released into the solution. This study provides a better understanding of the interactions between meta-autunite and microbes in conditions mimicking arid and semiarid subsurface environments of western U.S.

  12. Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

    SciTech Connect (OSTI)

    Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

    2015-12-02

    Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.

  13. Palladium nanoparticles produced by fermentatively cultivated bacteria as catalyst for diatrizoate removal with biogenic hydrogen

    SciTech Connect (OSTI)

    Hennebel, T.; Fitts, J.; Nevel, S. V.; Verschuere, S.; DeCorte, S.; DeGusseme, B.; Cuvelier, C.; vanderLelie, D.; Boon, N.; Verstraete, W.

    2011-05-17

    A new biological inspired method to produce nanopalladium is the precipitation of Pd on a bacterium, i.e., bio-Pd. This bio-Pd can be applied as catalyst in dehalogenation reactions. However, large amounts of hydrogen are required as electron donor in these reactions resulting in considerable costs. This study demonstrates how bacteria, cultivated under fermentative conditions, can be used to reductively precipitate bio-Pd catalysts and generate the electron donor hydrogen. In this way, one could avoid the costs coupled to hydrogen supply. The catalytic activities of Pd(0) nanoparticles produced by different strains of bacteria (bio-Pd) cultivated under fermentative conditions were compared in terms of their ability to dehalogenate the recalcitrant aqueous pollutants diatrizoate and trichloroethylene. While all of the fermentative bio-Pd preparations followed first order kinetics in the dehalogenation of diatrizoate, the catalytic activity differed systematically according to hydrogen production and starting Pd(II) concentration in solution. Batch reactors with nanoparticles formed by Citrobacter braakii showed the highest diatrizoate dehalogenation activity with first order constants of 0.45 {+-} 0.02 h{sup -1} and 5.58 {+-} 0.6 h{sup -1} in batches with initial concentrations of 10 and 50 mg L{sup -1} Pd, respectively. Nanoparticles on C. braakii, used in a membrane bioreactor treating influent containing 20 mg L{sup -1} diatrizoate, were capable of dehalogenating 22 mg diatrizoate mg{sup -1} Pd over a period of 19 days before bio-Pd catalytic activity was exhausted. This study demonstrates the possibility to use the combination of Pd(II), a carbon source and bacteria under fermentative conditions for the abatement of environmental halogenated contaminants.

  14. Towards an informative mutant phenotype for every bacterial gene

    SciTech Connect (OSTI)

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.

  15. Redirection of metabolism for hydrogen production

    SciTech Connect (OSTI)

    Harwood, Caroline S.

    2011-11-28

    This project is to develop and apply techniques in metabolic engineering to improve the biocatalytic potential of the bacterium Rhodopseudomonas palustris for nitrogenase-catalyzed hydrogen gas production. R. palustris, is an ideal platform to develop as a biocatalyst for hydrogen gas production because it is an extremely versatile microbe that produces copious amounts of hydrogen by drawing on abundant natural resources of sunlight and biomass. Anoxygenic photosynthetic bacteria, such as R. palustris, generate hydrogen and ammonia during a process known as biological nitrogen fixation. This reaction is catalyzed by the enzyme nitrogenase and normally consumes nitrogen gas, ATP and electrons. The applied use of nitrogenase for hydrogen production is attractive because hydrogen is an obligatory product of this enzyme and is formed as the only product when nitrogen gas is not supplied. Our challenge is to understand the systems biology of R. palustris sufficiently well to be able to engineer cells to produce hydrogen continuously, as fast as possible and with as high a conversion efficiency as possible of light and electron donating substrates. For many experiments we started with a strain of R. palustris that produces hydrogen constitutively under all growth conditions. We then identified metabolic pathways and enzymes important for removal of electrons from electron-donating organic compounds and for their delivery to nitrogenase in whole R. palustris cells. For this we developed and applied improved techniques in 13C metabolic flux analysis. We identified reactions that are important for generating electrons for nitrogenase and that are yield-limiting for hydrogen production. We then increased hydrogen production by blocking alternative electron-utilizing metabolic pathways by mutagenesis. In addition we found that use of non-growing cells as biocatalysts for hydrogen gas production is an attractive option, because cells divert all resources away from growth and to hydrogen. Also R. palustris cells remain viable in a non-growing state for long periods of time.

  16. Development of a Markerless Genetic Exchange System in Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

    SciTech Connect (OSTI)

    Keller, Kimberly L.; Bender, Kelly S.; Wall, Judy D.

    2009-07-21

    In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU); whereas, a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3')-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this inframe deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system, that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.

  17. The activity of CouR, a MarR family transcriptional regulator, is modulated through a novel molecular mechanism

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Otani, Hiroshi; Stogios, Peter J.; Xu, Xiaohui; Nocek, Boguslaw; Li, Shu -Nan; Savchenko, Alexei; Eltis, Lindsay D.

    2015-09-22

    CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl–CoA (Kd = 11 ± 1 μM). The presence of p-coumaroyl–CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl–CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR–p-coumaroyl–CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer and their CoAmore » moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl–CoA (Kd = 89 ± 6 μM). Altogether, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone.« less

  18. Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus

    SciTech Connect (OSTI)

    Pereira, Jose H.; Goh, Ee-Been; Keasling, Jay D.; Beller, Harry R.; Adams, Paul D.

    2012-10-01

    In an effort to better understand the control of the formation of branched fatty acids in Micrococcus luteus, the structure of ?-ketoacyl-ACP synthase III, which catalyzes the initial step of fatty-acid biosynthesis, has been determined. Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or ?-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.

  19. Microbial reduction of SO{sub 2} and NO{sub x} as a means of by-product recovery/disposal from regenerable processes for the desulfurization of flue gas. Technical progress report, September 11, 1992--December 11, 1992

    SciTech Connect (OSTI)

    Sublette, K.L.

    1992-12-31

    With the continual increase in the utilization of high sulfur and high nitrogen containing fossil fuels, the release of airborne pollutants into the environment has become a critical problem. The fuel sulfur is converted to SO{sub 2} during combustion. Fuel nitrogen and a fraction of the nitrogen from the combustion air are converted to nitric oxide and nitrogen dioxide, NO{sub x}. For the past five years Combustion Engineering (now Asea Brown Boveri or ABB) and, since 1986, the University of Tulsa (TU) have been investigating the oxidation of H{sub 2}S by the facultatively anaerobic and autotrophic bacterium Thiobacillus denitrificans and have developed a process, concept for the microbial removal of H{sub 2}S from a gas stream the simultaneous removal of SO{sub 2} and NO by D. desulfuricans and T. denitrificans co-cultures and cultures-in-series was demonstrated. These systems could not be sustained due to NO inhibition of D. desulfuricans. However, a preliminary economic analysis has shown that microbial reduction of SO{sub 2} to H{sub 2}S with subsequent conversion to elemental sulfur by the Claus process is both technically and economically feasible if a less expensive carbon and/or energy source can be found. It has also been demonstrated that T. denitrificans can be grown anaerobically on NO(g) as a terminal electron acceptor with reduction to elemental nitrogen. Microbial reduction of NO{sub x} is a viable process concept for the disposal of concentrated streams of NO{sub x} as may be produced by certain regenerable processes for the removal of SO{sub 2} and NO{sub x} from flue gas.

  20. Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

    SciTech Connect (OSTI)

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; Tomchick, Diana R.; Norgard, Michael V.

    2015-05-05

    The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.

  1. Detecting bacteria and Determining Their Susceptibility to Antibiotics by Stochastic Confinement in Nanoliter Droplets using Plug-Based Microfluidics

    SciTech Connect (OSTI)

    Boedicker, J.; Li, L; Kline, T; Ismagilov, R

    2008-01-01

    This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as stochastic confinement. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.

  2. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect (OSTI)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  3. Hydrogen-peroxide-induced oxidative stress responses in Desulfovibrio vulgaris Hildenborough

    SciTech Connect (OSTI)

    Zhou, A.; He, Z.; Redding-Johanson, A.M.; Mukhopadhyay, A.; Hemme, C.L.; Joachimiak, M.P.; Bender, K.S.; Keasling, J.D.; Stahl, D.A.; Fields, M.W.; Hazen, T.C.; Arkin, A.P.; Wall, J.D.; Zhou, J.; Luo, F.; Deng, Y.; He, Q.

    2010-07-01

    To understand how sulphate-reducing bacteria respond to oxidative stresses, the responses of Desulfovibrio vulgaris Hildenborough to H{sub 2}O{sub 2}-induced stresses were investigated with transcriptomic, proteomic and genetic approaches. H{sub 2}O{sub 2} and induced chemical species (e.g. polysulfide, ROS) and redox potential shift increased the expressions of the genes involved in detoxification, thioredoxin-dependent reduction system, protein and DNA repair, and decreased those involved in sulfate reduction, lactate oxidation and protein synthesis. A gene coexpression network analysis revealed complicated network interactions among differentially expressed genes, and suggested possible importance of several hypothetical genes in H{sub 2}O{sub 2} stress. Also, most of the genes in PerR and Fur regulons were highly induced, and the abundance of a Fur regulon protein increased. Mutant analysis suggested that PerR and Fur are functionally overlapped in response to stresses induced by H{sub 2}O{sub 2} and reaction products, and the upregulation of thioredoxin-dependent reduction genes was independent of PerR or Fur. It appears that induction of those stress response genes could contribute to the increased resistance of deletion mutants to H{sub 2}O{sub 2}-induced stresses. In addition, a conceptual cellular model of D. vulgaris responses to H{sub 2}O{sub 2} stress was constructed to illustrate that this bacterium may employ a complicated molecular mechanism to defend against the H{sub 2}O{sub 2}-induced stresses.

  4. Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Deng, Ying; Nagachar, Nivedita; Fang, Lin; Luan, Xin; Catchmark, Jeffrey M.; Tien, Ming; Kao, Teh -hui; Lai, Hsin -Chih

    2015-03-19

    Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To addressmore » this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.« less

  5. Tn-seq of Caulobacter crescentus under uranium stress reveals genes essential for detoxification and stress tolerance

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Yung, Mimi C.; Park, Dan M.; Overton, K. Wesley; Blow, Matthew J.; Hoover, Cindi A.; Smit, John R.; Murray, Sean R.; Ricci, Dante P.; Christen, Beat; Bowman, Grant R.; et al

    2015-07-20

    Ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. In order to gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFamore » and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. These results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus.« less

  6. Genome assortment, not serogroup, defines Vibrio cholerae pandemic strains

    SciTech Connect (OSTI)

    Brettin, Thomas S; Bruce, David C; Challacombe, Jean F; Detter, John C; Han, Cliff S; Munik, A C; Chertkov, Olga; Meincke, Linda; Saunders, Elizabeth; Choi, Seon Y; Haley, Bradd J; Taviani, Elisa; Jeon, Yoon - Seong; Kim, Dong Wook; Lee, Jae - Hak; Walters, Ronald A; Hug, Anwar; Colwell, Rita R

    2009-01-01

    Vibrio cholerae, the causative agent of cholera, is a bacterium autochthonous to the aquatic environment, and a serious public health threat. V. cholerae serogroup O1 is responsible for the previous two cholera pandemics, in which classical and El Tor biotypes were dominant in the 6th and the current 7th pandemics, respectively. Cholera researchers continually face newly emerging and re-emerging pathogenic clones carrying combinations of new serogroups as well as of phenotypic and genotypic properties. These genotype and phenotype changes have hampered control of the disease. Here we compare the complete genome sequences of 23 strains of V. cholerae isolated from a variety of sources and geographical locations over the past 98 years in an effort to elucidate the evolutionary mechanisms governing genetic diversity and genesis of new pathogenic clones. The genome-based phylogeny revealed 12 distinct V. cholerae phyletic lineages, of which one, designated the V. cholerae core genome (CG), comprises both O1 classical and EI Tor biotypes. All 7th pandemic clones share nearly identical gene content, i.e., the same genome backbone. The transition from 6th to 7th pandemic strains is defined here as a 'shift' between pathogenic clones belonging to the same O1 serogroup, but from significantly different phyletic lineages within the CG clade. In contrast, transition among clones during the present 7th pandemic period can be characterized as a 'drift' between clones, differentiated mainly by varying composition of laterally transferred genomic islands, resulting in emergence of variants, exemplified by V.cholerae serogroup O139 and V.cholerae O1 El Tor hybrid clones that produce cholera toxin of classical biotype. Based on the comprehensive comparative genomics presented in this study it is concluded that V. cholerae undergoes extensive genetic recombination via lateral gene transfer, and, therefore, genome assortment, not serogroup, should be used to define pathogenic V. cholerae clones.

  7. The activity of CouR, a MarR family transcriptional regulator, is modulated through a novel molecular mechanism

    SciTech Connect (OSTI)

    Otani, Hiroshi; Stogios, Peter J.; Xu, Xiaohui; Nocek, Boguslaw; Li, Shu -Nan; Savchenko, Alexei; Eltis, Lindsay D.

    2015-09-22

    CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl–CoA (Kd = 11 ± 1 μM). The presence of p-coumaroyl–CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl–CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR–p-coumaroyl–CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer and their CoA moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl–CoA (Kd = 89 ± 6 μM). Altogether, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone.

  8. Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

    SciTech Connect (OSTI)

    Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

    2010-06-01

    The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5’ end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

  9. Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

    2015-12-02

    Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins,more » and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.« less

  10. Towards an informative mutant phenotype for every bacterial gene

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

  11. Systems biology analysis of Zymomonas mobilis ZM4 ethanol stress responses

    SciTech Connect (OSTI)

    Yang, Shihui; Pan, Chongle; Tschaplinski, Timothy J; Hurst, Gregory {Greg} B; Engle, Nancy L; Zhou, Wen; Dam, Phuongan; Xu, Ying; Dice, Lezlee T; Davison, Brian H; Brown, Steven D

    2013-01-01

    Zymomonas mobilis ZM4 is a capable ethanogenic bacterium with high ethanol productivity and high level of ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of ethanol stress response have not been elucidated fully. In this study, ethanol stress responses were investigated using systems biology tools. Medium supplementation with an initial 47.3 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. Metabolomic profiling showed that ethanol-treated ZM4 cells accumulated greater amounts of glycerol during the entire fermentation process, which may indicate an important role for this metabolite. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 56% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. There were fewer genes significantly differentially expressed in the exponential phase compared to that of stationary phase and early stationary phase. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Correlations among the transcriptomics, proteomics and metabolism were examined and among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. This systems biology study elucidates key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress.

  12. Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

    SciTech Connect (OSTI)

    Sato, Chikara; Manaka, Sachie; Nakane, Daisuke; Nishiyama, Hidetoshi; Suga, Mitsuo; Nishizaka, Takayuki; Miyata, Makoto; Maruyama, Yuusuke

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Mycoplasma mobile was observed in buffer with the Atmospheric Scanning Electron Microscope. Black-Right-Pointing-Pointer Characteristic protein localizations were visualized using immuno-labeling. Black-Right-Pointing-Pointer M. mobile attached to sialic acid on the SiN film surface within minutes. Black-Right-Pointing-Pointer Cells were observed at low concentrations. Black-Right-Pointing-Pointer ASEM should promote study and early-stage diagnosis of mycoplasma. -- Abstract: Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3 {mu}m-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.

  13. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; Martí-Arbona, Ricardo

    2015-09-18

    FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

  14. The Structure and Function of an Arabinan-specific [alpha]-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

    SciTech Connect (OSTI)

    Cartmell, Alan; McKee, Lauren S.; Pena, Maria J.; Larsbrink, Johan; Brumer, Harry; Kaneko, Satoshi; Ichinose, Hitomi; Lewis, Richard J.; Vikso-Nielsen, Anders; Gilbert, Harry; Marles-Wright, Jon

    2012-03-26

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific {alpha}-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave {alpha}-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific {alpha}-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed {beta}-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for {alpha}-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  15. Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

    SciTech Connect (OSTI)

    Gao, Bin; Zhu, Shunyi

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Al-M is an engineered fungal defensin with the n-loop of an insect defensin. Black-Right-Pointing-Pointer Al-M adopts a native defensin-like structure with high antibacterial potency. Black-Right-Pointing-Pointer Al-M kills bacteria through a membrane disruptive mechanism. Black-Right-Pointing-Pointer This work sheds light on the functional evolution of CS{alpha}{beta}-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized {alpha}-helical and {beta}-sheet (CS{alpha}{beta}) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 {mu}M. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

  16. Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility

    SciTech Connect (OSTI)

    Serer, Mara I.; Bonomi, Hernn R.; Guimares, Beatriz G.; Rossi, Rolando C.; Goldbaum, Fernando A.; Klinke, Sebastin

    2014-05-01

    This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed. Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C{sub 3} symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.

  17. Genomic analyses of bacterial porin-cytochrome gene clusters

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Shi, Liang; Fredrickson, James K.; Zachara, John M.

    2014-11-26

    In this study, the porin-cytochrome (Pcc) protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III) by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c type cytochrome (c-Cyt) and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters) of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteriamore » from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr) gene clusters of other Fe(III)-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III) and Mn(IV) oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III) and Mn(IV) oxides.« less

  18. A Small Company Partnership with Global Impact: Portable, Affordable, and Reliable Anthrax Testing

    Broader source: Energy.gov [DOE]

    Anthrax, an infectious disease caused by the bacterium Bacillus anthracic, poses a significant threat to U.S. national security as demonstrated by the 2001 terrorist attacks that targeted the U.S. Postal Service and Hart Senate Office Building in Washington, DC. Sandia National Laboratories developed an anthrax detection sensor for low-resource environments called the Anthrax Detection Cartridge. The portable device, developed from a Laboratory Directed Research and Development project, is inexpensive and requires no power to run and minimal training to operate. It quickly provides highly reliable anthrax detection in controlled environments, rivaling the selectivity of rigorous laboratory analysis. Winner of a 2014 R&D 100 Award, the Anthrax Detection Cartridge is a self-contained, credit-card sized test system that cultures a sample in a patent-pending amplification chamber using selective growth media. Once a sample is inserted, patent-pending magnetically operated valves advance it from stage to stage to complete the testing process. The device uses a lateral flow assay (LFA) to determine if the sample is dangerous anthrax, and then treats the sample with disinfectant. Anthrax outbreaks are common in livestock and pose significant risks to animal and public health. Diagnosis currently often requires isolation and analysis of the organism within a laboratory. These resources are often absent or difficult to obtain in rural or poorer areas. With minor modifications, simply swapping out the selective growth medium and LFA strip, the cartridges can be adapted to detect other bacteria, such as salmonella in agricultural settings, as well as bacteria of medical interest. Aquila, a woman-owned small business based in New Mexico that specializes in the design and manufacture of technologies and services for nuclear security and international safeguards, is licensing the Anthrax Detection Cartridge technology and plans to manufacture the device. Company officials see a potential market among government and commercial customers. Company officials praised the technical support they received from Sandia as well as the smooth and rapid licensing process. An Umbrella Cooperative Research and Development Agreement (CRADA) between Aquila and Sandia should result in more collaboration on this and other joint projects. Sandia is continuing to refine the technology and adapt it for other markets interested in rapid detection of biological hazards.

  19. Genomic analyses of bacterial porin-cytochrome gene clusters

    SciTech Connect (OSTI)

    Shi, Liang; Fredrickson, James K.; Zachara, John M.

    2014-11-26

    In this study, the porin-cytochrome (Pcc) protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III) by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c type cytochrome (c-Cyt) and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters) of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteria from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr) gene clusters of other Fe(III)-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III) and Mn(IV) oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III) and Mn(IV) oxides.

  20. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

    SciTech Connect (OSTI)

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth; Arkin, Adam P.; Deutschbauer, Adam

    2015-05-12

    Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

  1. Epidemiology and Control of Infectious Diseases of Salmonids in the Columbia River Basin, 1987 Annual Report.

    SciTech Connect (OSTI)

    Fryer, John L.

    1989-01-01

    The Department of Microbiology at Oregon State University with funding from the Bonneville Power Administration has been conducting a study concerning the epidemiology and control of three fish pathogens which cause major disease problems in salmonids of the Columbia River basin. The pathogens studied include Cera to myxa Shasta, the myxosporean parasite which causes ceratomyxosis; Renibacterium salmoninarum, the bacterium which is the etiological agent of bacterial kidney disease; and the rhabdovirus which causes infectious hematopoietic necrosis (IHN). During this project, the host and geographic range of C. Shasta have been more precisely determined and the known geographic range has been significantly expanded. The effects of the parasite on fish migrating through the Columbia River and on their introduction into salt water have been examined. Similar studies have been conducted with R. salmoninarum and it has been shown that bacterial kidney disease occurs at all life stages of salmonids and is responsible for mortality in both fresh and salt water. It has also been demonstrated that different isolates of R. salmoninarum have different antigenic composition. Results of demonstration projects designed to control IHN by using UV treated water for early rearing of salmonid fry were equivocal. The scope of the project was considerably narrowed and focused during the past two years The project has concentrated on a study concerning the biology of C. Shasta and the identification of potential chemotherapeutants for control of bacterial kidney disease. The emphasis of work on C. Shasta has been its pathogenesis. This aspect of the parasite has been investigated using histopathologic and immunologic methodology. Mode of transmission, the nature of the infectious stage, and potential intermediate hosts of the parasite have also been areas of active research. Classes of chemotherapeutants with the highest potential for efficacy against R. salmoninarum have been identified through literature searches and consultation with pharmacologists. Experimental drugs have been requested and received from several pharmaceutical manufacturers. The in vitro sensitivity of R. salmoninarum and other selected fish pathogens to more than 100 antimicrobial compounds has been tested. The project is related to measure 704(h)(2)(d) of the Columbia River Basin Fish and Wildlife Program. The results will contribute to fish health which will directly contribute to the protection of fish.

  2. Intercellular Genomics of Subsurface Microbial Colonies

    SciTech Connect (OSTI)

    Ortoleva, Peter; Tuncay, Kagan; Gannon, Dennis; Meile, Christof

    2007-02-14

    This report summarizes progress in the second year of this project. The objective is to develop methods and software to predict the spatial configuration, properties and temporal evolution of microbial colonies in the subsurface. To accomplish this, we integrate models of intracellular processes, cell-host medium exchange and reaction-transport dynamics on the colony scale. At the conclusion of the project, we aim to have the foundations of a predictive mathematical model and software that captures the three scales of these systems – the intracellular, pore, and colony wide spatial scales. In the second year of the project, we refined our transcriptional regulatory network discovery (TRND) approach that utilizes gene expression data along with phylogenic similarity and gene ontology analyses and applied it successfully to E.coli, human B cells, and Geobacter sulfurreducens. We have developed a new Web interface, GeoGen, which is tailored to the reconstruction of microbial TRNs and solely focuses on Geobacter as one of DOE’s high priority microbes. Our developments are designed such that the frameworks for the TRND and GeoGen can readily be used for other microbes of interest to the DOE. In the context of modeling a single bacterium, we are actively pursuing both steady-state and kinetic approaches. The steady-state approach is based on a flux balance that uses maximizing biomass growth rate as its objective, subjected to various biochemical constraints, for the optimal values of reaction rates and uptake/release of metabolites. For the kinetic approach, we use Karyote, a rigorous cell model developed by us for an earlier DOE grant and the DARPA BioSPICE Project. We are also investigating the interplay between bacterial colonies and environment at both pore and macroscopic scales. The pore scale models use detailed representations for realistic porous media accounting for the distribution of grain size whereas the macroscopic models employ the Darcy-type flow equations and up-scaled advective-diffusive transport equations for chemical species. We are rigorously testing the relationship between these two scales by evaluating macroscopic parameters using the volume averaging methodology applied to pore scale model results.

  3. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

    DOE Public Access Gateway for Energy & Science Beta (PAGES Beta)

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth; Arkin, Adam P.; et al

    2015-05-12

    Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

  4. Engineering and Coordination of Regulatory Networks and Intracellular Complexes to Maximize Hydrogen Production by Phototrophic Microorganisms

    SciTech Connect (OSTI)

    James C. Liao

    2012-05-22

    This project is a collaboration with F. R. Tabita of Ohio State. Our major goal is to understand the factors and regulatory mechanisms that influence hydrogen production. The organisms to be utilized in this study, phototrophic microorganisms, in particular nonsulfur purple (NSP) bacteria, catalyze many significant processes including the assimilation of carbon dioxide into organic carbon, nitrogen fixation, sulfur oxidation, aromatic acid degradation, and hydrogen oxidation/evolution. Our part of the project was to develop a modeling technique to investigate the metabolic network in connection to hydrogen production and regulation. Organisms must balance the pathways that generate and consume reducing power in order to maintain redox homeostasis to achieve growth. Maintaining this homeostasis in the nonsulfur purple photosynthetic bacteria is a complex feat with many avenues that can lead to balance, as these organisms possess versatile metabolic capabilities including anoxygenic photosynthesis, aerobic or anaerobic respiration, and fermentation. Growth is achieved by using H{sub 2} as an electron donor and CO{sub 2} as a carbon source during photoautotrophic and chemoautotrophic growth, where CO{sub 2} is fixed via the Calvin-Benson-Bassham (CBB) cycle. Photoheterotrophic growth can also occur when alternative organic carbon compounds are utilized as both the carbon source and electron donor. Regardless of the growth mode, excess reducing equivalents generated as a result of oxidative processes, must be transferred to terminal electron acceptors, thus insuring that redox homeostasis is maintained in the cell. Possible terminal acceptors include O{sub 2}, CO{sub 2}, organic carbon, or various oxyanions. Cells possess regulatory mechanisms to balance the activity of the pathways which supply energy, such as photosynthesis, and those that consume energy, such as CO{sub 2} assimilation or N{sub 2} fixation. The major route for CO{sub 2} assimilation is the CBB reductive pentose phosphate pathway, whose key enzyme is ribulose 1,5-biphosphate carboxylase/oxygenase (RubisCO). In addition to providing virtually all cellular carbon during autotrophic metabolism, RubisCO-mediated CO{sub 2} assimilation is also very important for nonsulfur purple photosynthetic bacteria under photoheterotrophic growth conditions since CO{sub 2} becomes the major electron sink under these conditions. In this work, Ensemble Modeling (EM) was developed to examine the behavior of CBB-compromised RubisCO knockout mutant strains of the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides. Mathematical models of metabolism can be a great aid in studying the effects of large perturbations to the system, such as the inactivation of RubisCO. Due to the complex and highly-interconnected nature of these networks, it is not a trivial process to understand what the effect of perturbations to the metabolic network will be, or vice versa, what enzymatic perturbations are necessary to yield a desired effect. Flux distribution is controlled by multiple enzymes in the network, often indirectly linked to the pathways of interest. Further, depending on the state of the cell and the environmental conditions, the effect of a perturbation may center around how it effects the carbon flow in the network, the balancing of cofactors, or both. Thus, it is desirable to develop mathematical models to describe, understand, and predict network behavior. Through the development of such models, one may gain the ability to generate a set of testable hypotheses for system behavior.

  5. Molecular Characterization of Bacterial Respiration on Minerals

    SciTech Connect (OSTI)

    Blake, Robert C.

    2013-04-26

    The overall aim of this project was to contribute to our fundamental understanding of proteins and biological processes under extreme environmental conditions. We sought to define the biochemical and physiological mechanisms that underlie biodegradative and other cellular processes in normal, extreme, and engineered environments. Toward that end, we sought to understand the substrate oxidation pathways, the electron transport mechanisms, and the modes of energy conservation employed during respiration by bacteria on soluble iron and insoluble sulfide minerals. In accordance with these general aims, the specific aims were two-fold: To identify, separate, and characterize the extracellular biomolecules necessary for aerobic respiration on iron under strongly acidic conditions; and to elucidate the molecular principles whereby these bacteria recognize and adhere to their insoluble mineral substrates under harsh environmental conditions. The results of these studies were described in a total of nineteen manuscripts. Highlights include the following: 1. The complete genome of Acidithiobacillus ferrooxidans ATCC 23270 (type strain) was sequenced in collaboration with the DOE Joint Genome Institute; 2. Genomic and mass spectrometry-based proteomic methods were used to evaluate gene expression and in situ microbial activity in a low-complexity natural acid mine drainage microbial biofilm community. This was the first effort to successfully analyze a natural community using these techniques; 3. Detailed functional and structural studies were conducted on rusticyanin, an acid-stable electron transfer protein purified from cell-free extracts of At. ferrooxidans. The three-dimensional structure of reduced rusticyanin was determined from a combination of homonuclear proton and heteronuclear 15N- and 13C-edited NMR spectra. Concomitantly, the three-dimensional structure of oxidized rusticyanin was determined by X-ray crystallography to a resolution of 1.9 A by multiwavelength anomalous dispersion (MAD) phasing; 4. An acid-stable red cytochrome with a novel absorbance peak at 579 nm was purified from cell-free extracts of L. ferriphilum. Functional studies demonstrated that this cytochrome was an important component of the aerobic iron respiratory chain in this organism; 5. The specific adhesion of At. ferrooxidans to pyrite is mediated by an extracellular protein that was identified as aporusticyanin. The adhesion of At. ferrooxidans to minerals was characterized by high affinity binding that exhibited a high specificity for pyrite over other sulfide minerals. The principal biopolymer involved in this high-affinity adhesion to pyrite was isolated by mineral affinity chromatography and identified as aporusticyanin. The adhesion of purified aporusticyanin to minerals was observed to adhere to different mineral with a pattern of reactivity identical to that observed with the intact bacterium. Further, preincubation of pyrite with excess exogenous aporusticyanin served to inhibit the adherence of intact cells to the surface of the mineral, indicating that the protein and the cells adhered to the pyrite in a mutually exclusive manner. Taken together, these observations support a model where aporusticyanin located on the surface of the bacterial cell acts as a mineral-specific receptor for the initial adherence of At. ferrooxidans to solid pyrite; 6. The specific adhesion of L. ferriphilum to pyrite was mediated by a different acid-stable extracellular protein than aporusticyanin; and 7. A prototype integrating cavity absorption meter (ICAM) was assembled to determine whether this novel spectrophotometer could be used to study cellular respiration in situ.

  6. Technetium Reduction and Permanent Sequestration by Abiotic and Biotic Formation of Low-Solubility Sulfide Mineral Phases

    SciTech Connect (OSTI)

    Tratnyek, Paul G.; Tebo, Bradley M.; Fan, Dimin; Anitori, Roberto; Szecsody, Jim; Jansik, Danielle

    2015-11-14

    One way to minimize the mobility of the TcVII oxyanion pertechnetate (TcO4-) is to effect reduction under sulfidogenic conditions (generated abiotically by Fe0 or biotically) to form TcSx, which is significantly slower to oxidize than TcIVO2. In sediment systems, TcSx and other precipitates may oxidize more slowly due to oxygen diffusion limitations to these low permeability precipitate zones. In addition, the TcO4- reduction rate may be more rapid in the presence of sediment because of additional reductive surface phases. This project aims to provide a fundamental understanding of the feasibility of immobilization of TcO4- as TcSx in the vadose zone or groundwater by application nano zero-valent iron (nZVI), and sulfide or sulfate. Biotic batch experiments have used the sulfate-reducing bacterium (SRB) Desulfotomaculum reducens. The iron sulfide mineral mackinawite was generated under these conditions, while vivianite was formed in nZVI only controls. The sulfide/bacteria-containing system consistently reduced aqueous pertechnetate rapidly (> 95% in the first hour), a rate similar to that for the sulfide-free, nZVI only system. Reduced Tc (aged for 3 months) generated in both SRB/nZVI systems was highly resistant to reoxidation. In reduced samples, Tc was found associated with solid phases containing Fe and S (D. reducens/nZVI) or Fe (nZVI only). Experiments using D. reducens without nZVI provided some additional insights. Firstly, stationary phase cultures were able to slowly reduce pertechnetate. Secondly, addition of pertechnetate at the beginning of cell growth (lag phase) resulted in a faster rate of Tc reduction, possibly indicating a direct (e.g. enzymatic) role for D. reducens in Tc reduction. Abiotic batch experiments were conducted with Na2S as the sulfide source. Pertechnetate reduction was rapid in the presence of sulfide and nZVI, although the rate was suppressed at the higher S/Fe ratios tested. This suppression appeared to be due to the formation of Tc-containing colloids. As with the biotic experiments, pertechnetate reduced under sulfidic conditions was highly resistant to reoxidation. The microscopic morphology of abiotically-transformed nZVI particles varied significantly with those in the biotic experiment, although mackinawite was formed in both systems (as indicated by μXRD and Mössbauer spectroscopy). Preliminary XAS analysis pointed to a mixture of Tc-O and Tc-S binding in the abiotic sulfide/nZVI system, while the major reduced solids under non-sulfidic conditions were TcO2•nH2O. The presence of sediment and advective flow to the TcO4-/nZVI/sulfide system results in additional processes occurring. Although the natural Hanford sediment used has sufficient available ferrous iron to slowly reduce TcO4-, under anaerobic conditions, that rate is orders of magnitude slower than reduction by nZVI/sulfide. Batch and 1-D column experiments showed that the TcO4- reduction rate increased with the sediment surface area (with the same nZVI mass). As in batch systems, column studies showed that the presence of sulfide with TcO4- at low (2-5 mM) concentrations increased the TcO4- reduction rate and high (10-30 mM) sulfide decreased the rate. This change is attributed to the formation of sulfide precipitates on the nZVI and sediment surfaces. Injection of low and high sulfide (i.e. pretreatment) prior to TcO4-/sulfide injection also greatly decreased the TcO4- reduction rate, likely decreasing the generation of ferrous iron from the nZVI. Although the high sulfide systems have slower Tc reduction rates, 190 times more Tc mass precipitated than in the low sulfide systems and the highest fraction of Tc mass remained immobilized.

  7. Development of a Low Input and sustainable Switchgrass Feedstock Production System Utilizing Beneficial Bacterial Endophytes

    SciTech Connect (OSTI)

    Mei, Chuansheng; Nowak, Jerzy; Seiler, John

    2014-10-24

    Switchgrass represents a promising feedstock crop for US energy sustainability. However, its broad utilization for bioenergy requires improvements of biomass yields and stress tolerance. In this DOE funded project, we have been working on harnessing beneficial bacterial endophytes to enhance switchgrass performance and to develop a low input feedstock production system for marginal lands that do not compete with the production of food crops. We have demonstrated that one of most promising plant growth-promoting bacterial endophytes, Burkholderia phytofirmans strain PsJN, is able to colonize roots and significantly promote growth of switchgrass cv. Alamo under in vitro, growth chamber, greenhouse, as well as field conditions. Furthermore, PsJN bacterization improved growth and development of switchgrass seedlings, significantly stimulated plant root and shoot growth, and tiller number in the field, and enhanced biomass accumulation on both poor (p<0.001) and rich (p<0.05) soils, with more effective stimulation of plant growth in low fertility soil. Plant physiology measurements showed that PsJN inoculated Alamo had consistently lower transpiration, lower stomatal conductance, and higher water use efficiency in greenhouse conditions. These physiological changes may significantly contribute to the recorded growth enhancement. PsJN inoculation rapidly results in an increase in photosynthetic rates which contributes to the advanced growth and development. Some evidence suggests that this initial growth advantage decreases with time when resources are not limited such as in greenhouse studies. Additionally, better drought resistance and drought hardening were observed in PsJN inoculated switchgrass. Using the DOE-funded switchgrass EST microarray, in a collaboration with the Genomics Core Facility at the Noble Foundation, we have determined gene expression profile changes in both responsive switchgrass cv. Alamo and non-responsive cv. Cave-in-Rock (CR) following PsJN bacterization. With the MapMan software to analyze microarray data, the number of up- and down-regulated probes was calculated. The number of up-regulated probes in Alamo was 26, 14, 14, and 12% at 0.5, 2, 4 and 8 days after inoculation (DAI) with PsJN, respectively while the corresponding number in CR was 24, 22, 21, and 19%, respectively. In both cultivars, the largest number of up-regulated probes occurred at 0.5 DAI. Noticeable differences throughout the timeframe between Alamo and CR were that the number was dramatically decreased to half (12%) in Alamo but remained high in CR (approximately 20%). The number of down regulated genes demonstrated different trends in Alamo and CR. Alamo had an increasing trend from 9% at 0.5 DAI to 11, 17, and 28% at 2, 4, and 8 DAI, respectively. However, CR had 13% at 0.5 and 2 DAI, and declined to 10% at 4 and 8 DAI. With the aid of MapMan and PageMan, we mapped the response of the ID probes to the observed major gene regulatory network and major biosynthetic pathway changes associated with the beneficial bacterial endophyte infection, colonization, and early growth promotion process. We found significant differences in gene expression patterns between responsive and non-responsive cultivars in many pathways, including redox state regulation, signaling, proteolysis, transcription factors, as well as hormone (SA and JA in particular)-associated pathways. Form microarray data, a total of 50 key genes have been verified using qPCR. Ten of these genes were chosen for further functional study via either overexpression and/or RNAi knockout technologies. These genes were calmodulin-related calcium sensor protein (CAM), glutathione S-transferase (GST), histidine-containing phosphotransfer protein (H-221), 3 different zinc finger proteins (ZF-371, ZF131 and ZF242), EF hand transcription factor (EF-622), peroxidase, cellulose synthase catalytic submit A2 (CESA2), and Aux/IAA family. A total of 8 overexpression and 5 RNAi transgenic plants have been regenerated, and their gene expression levels determined using qPCR. Consequently high, medium and low expression lines were propagated in vitro for gene function study. When adequate numbers of individual transgenic lines were obtained, they were challenged with PsJN to see if PsJN promotes or inhibits growth of transgenic plants. Our results demonstrated that EF-622 overexpression, ZF-371, GST, H-221 and CAM RNAi transgenic lines lost responses to PsJN, i.e. PsJN had no growth promotive effects on these transgenic plants. Further study needs to be done to characterize this loss of responsiveness to PsJN. During this funding period, we have done more work related to this funded project and established collaborations with other institutions and obtained some interesting results, building a foundation for further research projects. For example, we isolated a naturally-occurring bacterium from surface-sterilized switchgrass seeds, identified as a unique Panteoa agglomerans species, and named strain PaKM. PaKM has been proved to be an efficient growth promoter of switchgrass over a broad spectrum of genotypes and has potential in applications with low input and sustainable production systems on marginal lands. In collaboration with Dr. Shuijin Hu (North Carolina State University), we conducted experiments on how endophyte-inoculated switchgrass affects soil N and P availability and the number of AMF in roots. Our preliminary results showed that PsJN increased AMF infection of switchgrass roots, and enhanced soil N availability and soil N mineralization on a low nutrient field. Further study of this phenomenon on different soils, over longer time periods, is needed to assess its potential impact on the productivity and longevity of switchgrass stands.