16 Search Results

Economically viable production of cellulosic biofuels requires operation at high solids loadings—on the order of 15 wt%. To this end we characterize Nature’s ability to deconstruct and utilize mid-season switchgrass at increasing solid loadings using an anaerobic methanogenic microbiome. This community exhibits undiminished fractional carbohydrate solubilization at loadings ranging from 30 g/L to 150 g/L. Metaproteomic interrogation reveals marked increases in the abundance of specific carbohydrate-active enzyme classes. Significant enrichment of auxiliary activity family 6 enzymes at higher solids suggests a role for Fenton chemistry. Stress-response proteins accompanying these reactions are similarly upregulated at higher solids, as are β-glucosidases, xylosidases,more » carbohydrate-debranching, and pectin-acting enzymes—all of which indicate that removal of deconstruction inhibitors is important for observed undiminished solubilization. Our work provides insights into the mechanisms by which natural microbiomes effectively deconstruct and utilize lignocellulose at high solids loadings, informing the future development of defined cultures for efficient bioconversion.« less

There are known associations between opioids, obesity, and the gut microbiome, but the molecular connection/mediation of these relationships is not understood. To better clarify the interplay of physiological, genetic, and microbial factors, this study investigated the microbiome and host inflammatory responses to chronic opioid administration in genetically obese, diet-induced obese, and lean mice. Samples of feces, urine, colon tissue, and plasma were analyzed using targeted LC-MS/MS quantification of metabolites, immunoassays of inflammatory cytokine levels, genome-resolved metagenomics, and metaproteomics. Genetic obesity, diet-induced obesity, and morphine treatment in lean mice each showed increases in distinct inflammatory cytokines. Metagenomic assembly and binning uncoveredmore » over 400 novel gut bacterial genomes and species. Morphine administration impacted the microbiome’s composition and function, with the strongest effect observed in lean mice. This microbiome effect was less pronounced than either diet or genetically driven obesity. Based on inferred microbial physiology from the metaproteome datasets, a high-fat diet transitioned constituent microbes away from harvesting diet-derived nutrients and towards nutrients present in the host mucosal layer. Considered together, these results identified novel host-dependent phenotypes, differentiated the effects of genetic obesity versus diet induced obesity on gut microbiome composition and function, and showed that chronic morphine administration altered the gut microbiome.« less

Soil microbiomes harbour unparalleled functional and phylogenetic diversity. However, extracting isolates with a targeted function from complex microbiomes is not straightforward, particularly if the associated phenotype does not lend itself to high-throughput screening. Here, we tackle the methylation of arsenic (As) in anoxic soils. As methylation was proposed to be catalysed by sulfate-reducing bacteria. However, to date, there are no available anaerobic isolates capable of As methylation, whether sulfate-reducing or otherwise. The isolation of such a microorganism has been thwarted by the fact that the anaerobic bacteria harbouring a functional arsenite S-adenosylmethionine methyltransferase (ArsM) tested to date did not methylatemore » As in pure culture. Additionally, fortuitous As methylation can result from the release of non-specific methyltransferases upon lysis. Thus, we combined metagenomics, metatranscriptomics, and metaproteomics to identify the microorganisms actively methylating As in anoxic soil-derived microbial cultures. Based on the metagenome-assembled genomes of microorganisms expressing ArsM, we isolated Paraclostridium sp. strain EML, which was confirmed to actively methylate As anaerobically. This work is an example of the application of meta-omics to the isolation of elusive microorganisms.« less

Abstract Background Mass spectrometry-based proteomics can identify and quantify thousands of proteins from individual microbial species, but a significant percentage of these proteins are unannotated and hence classified as proteins of unknown function (PUFs). Due to the difficulty in extracting meaningful metabolic information, PUFs are often overlooked or discarded during data analysis, even though they might be critically important in functional activities, in particular for metabolic engineering research. Results We optimized and employed a pipeline integrating various “guilt-by-association” (GBA) metrics, including differential expression and co-expression analyses of high-throughput mass spectrometry proteome data and phylogenetic coevolution analysis, and sequence homology-based approachesmore » to determine putative functions for PUFs in Clostridium thermocellum . Our various analyses provided putative functional information for over 95% of the PUFs detected by mass spectrometry in a wild-type and/or an engineered strain of C. thermocellum . In particular, we validated a predicted acyltransferase PUF (WP_003519433.1) with functional activity towards 2-phenylethyl alcohol, consistent with our GBA and sequence homology-based predictions. Conclusions This work demonstrates the value of leveraging sequence homology-based annotations with empirical evidence based on the concept of GBA to broadly predict putative functions for PUFs, opening avenues to further interrogation via targeted experiments.« less

Rising temperatures in the Arctic have led to the thawing of tundra soils, which is rapidly changing terrain, hydrology, and plant and microbial communities, causing hotspots of biogeochemical activity across the landscape. Despite this, little is known about how nutrient-rich low molecular weight dissolved organic matter (LMW DOM) varies within and across tundra ecosystems. Using a high-resolution nano-liquid chromatography-mass spectrometry (LC/MS) approach, we characterized the composition and availability of LMW DOM from high-centered polygons (HCP) and low-centered polygons (LCP) with Eriophorum angustifolium or Carex aquatilis as the dominant vegetation. Over 3000 unique features (i.e., discrete mass/charge ions) were detected; 521more » were identified as differentially abundant between polygonal types and 217 were putatively annotated using high mass accuracy MS data. While polygon type was a strong predictor of LMW DOM composition and availability, vegetation and soil depth were also important drivers. Extensive evidence was found for enhanced microbial processing at the LCP sites, which were dominated by Carex plant species. We detected significant differences between polygon types with varying aboveground landscape features or properties, and hotspots of biogeochemical activity, indicating LMW DOM, as quantified by untargeted exometabolomics, provides a window into the dynamic complex interactions between landscape topography, vegetation, and organic matter cycling in Arctic polygonal tundra soils.« less

Abstract Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum onmore » corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners— Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum —exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.« less

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Resultsmore » revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.« less

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alterationmore » in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.« less

Paenibacillus polymyxa is a Gram-positive bacterium commonly found associated with plant roots. P. polymyxa can exhibit two forms of flagellar motility: swimming in liquid culture and swarming on a surface. Here, swimming cells were compared to swarming cells using an integrated proteomic and lipidomic approach, yielding information about how lipid modifications and protein/enzyme pathways are tailored for these specific phenotypes. Observed differences in both phospholipid composition and metabolism between the two conditions suggest membrane remodeling in response to the surrounding environment. Key enzymes involved in glycerophospholipid metabolism were abundant in swimming bacteria, while enzymes associated with glycerol-3-phosphate metabolism were moremore » abundant in swarming bacteria. Several glycoside hydrolases were either unique to or more abundant during swarming. This likely reflects the degradation of their own exopolysaccharides to both enhance swarming and supply the necessary chemical energy to compensate for increased flagellar synthesis. The observed upregulation of biosynthetic gene clusters (polyketides, lantibiotics, and surfactin) in swarming bacteria suggest the importance of signaling, antimicrobial activity, and surfactin production during this mode of motility – the latter of which is confirmed via RT-PCR.« less

Small peptides that are proteolytic cleavage products (PCPs) of less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective enrichment and subsequent characterization by mass spectrometry–based sequencing offers the greatest potential for their comprehensive characterization, however qualitative and quantitative performance metrics are rarely captured. Herein, we addressed this need by benchmarking the performance of an enrichment strategy, optimized specifically for small PCPs, using state-of-the-art de novo–assisted peptide sequencing. As amore » case study, we implemented this approach to identify PCPs from different root and foliar tissues of the hybrid poplar Populus × canescens 717-1B4 in interaction with the ectomycorrhizal basidiomycete Laccaria bicolor. In total, we identified 1,660 and 2,870 Populus and L. bicolor unique PCPs, respectively. Qualitative results supported the identification of well-known PCPs, like the mature form of the photosystem II complex 5-kDa protein (approximately 3 kDa). A total of 157 PCPs were determined to be significantly more abundant in root tips with established ectomycorrhiza when compared with root tips without established ectomycorrhiza and extramatrical mycelium of L. bicolor. These PCPs mapped to 64 Populus proteins and 69 L. bicolor proteins in our database, with several of them previously implicated in biologically relevant associations between plant and fungus.« less