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Title: "Final Report for Grant No. DE-FG02-97ER62492 "Engineering Deinococcus radiodurans for Metal Remediation in Radioactive Mixed Waste Sites"

Abstract

The groundwater and sediments of numerous U. S. Department of Energy (DOE) field sites are contaminated with mixtures of heavy metals (e.g., Hg, Cr, Pd) and radionuclides (e.g., U, Tc), as well as the fuel hydrocarbons benzene, toluene, ethylbenzene and xylenes (BTEX); chlorinated hydrocarbons, such as trichloroethylene (TCE); and polychlorinated biphenyls (PCBs). The remediation of such mixed wastes constitutes an immediate and complex waste management challenge for DOE, particularly in light of the costliness and limited efficacy of current physical and chemical strategies for treating mixed wastes. In situ bioremediation via natural microbial processes (e.g., metal reduction) remains a potent, potentially cost-effective approach to the reductive immobilization or detoxification of environmental contaminants. Seventy million cubic meters of soil and three trillion liters of groundwater have been contaminated by leaking radioactive waste generated in the United States during the Cold War. A cleanup technology is being developed based on the extremely radiation resistant bacterium Deinococcus radiodurans. Our recent isolation and characterization of D. radiodurans from a variety of DOE environments, including highly radioactive sediments beneath one of the leaking tanks (SX-108) at the Hanford Site in south-central Washington state, underscores the potential for this species to survive in such extreme environments.more » Research aimed at developing D. radiodurans for metal remediation in radioactive waste sites was started by this group in September 1997 with support from DOE NABIR grant DE-FG02-97ER62492. Our grant was renewed for the period 2000-2003, which includes work on the thermophilic radiation resistant bacterium Deinococcus geothermalis. Work funded by the existing grant contributed to 18 papers in the period 1997-2004 on the fundamental biology of D. radiodurans and its design for bioremediation of radioactive waste environments. Our progress since September 2000 closely matches the Aims proposed in our second NABIR application and is summarized as follows. We have further refined expression vectors for D. radiodurans and successfully tested engineered strains in natural DOE sediment and groundwater samples. Further, we have shown that D. geothermalis is transformable with plasmids and integration vectors designed for D. radiodurans. This was demonstrated by engineering Hg(II)-resistant D. geothermalis strains capable of reducing Hg(II) at elevated temperatures and under chronic irradiation. Additionally, we showed that D. geothermalis, like D. radiodurans, is naturally capable of reducing U(VI), Cr(VI), and Fe(III). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55 C, of which there are many examples. Our annotation of the D. radiodurans genome has been an important guide throughout this project period and continues to be a source of inspiration in the development of new genetic technologies dedicated to this bacterium. For example, our genome analyses have enabled us to achieve engineering goals that were unattainable in our first NABIR project (1997-2000), where uncertainties relating to its metabolic configuration prevented efforts to expand its metabolic capabilities. As just one example, we showed that D. radiodurans has a functioning tricarboxylic acid (TCA) cycle glyoxylate bypass which could be integrated with toluene oxidation. And, we successfully engineered D. radiodurans to derive carbon and energy from complete toluene mineralization and showed that toluene oxidation can be coupled to cellular biosynthesis, survival, as well as its native and engineered metal reducing capabilities. We have also constructed a whole genome microarray for D. radiodurans covering {approx}94% of its predicted genes and have successfully used the array to examine the response of cells to radiation and other DOE relevant conditions. Similarly, we have used high throughput proteomic approaches to examine how D. radiodurans responds to a variety of stress conditions. These studies have validated our annotation and are facilitating analysis of its metabolism, resistance, and metal reduction pathways.« less

Authors:
Publication Date:
Research Org.:
The Henry M. Jackson Foundation
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
837824
Report Number(s):
DOE/ER/62492-1
TRN: US0701851
DOE Contract Number:
FG02-97ER62492
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
12 MANAGEMENT OF RADIOACTIVE WASTES, AND NON-RADIOACTIVE WASTES FROM NUCLEAR FACILITIES; 54 ENVIRONMENTAL SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; BENZENE; BIOLOGY; BIOREMEDIATION; BIOSYNTHESIS; CHRONIC IRRADIATION; GENETICS; HEAVY METALS; HYDROCARBONS; MINERALIZATION; ORGANIC CHLORINE COMPOUNDS; OXIDATION; POLYCHLORINATED BIPHENYLS; RADIOACTIVE WASTES; RADIOISOTOPES; TOLUENE; WASTE MANAGEMENT; XYLENES; Deinococcus; Bioremediation; Radionuclides; Heavy Metals; Toxic Organic Compounds; Radiation

Citation Formats

Michael J. Daly, Ph.D.. "Final Report for Grant No. DE-FG02-97ER62492 "Engineering Deinococcus radiodurans for Metal Remediation in Radioactive Mixed Waste Sites". United States: N. p., 2005. Web. doi:10.2172/837824.
Michael J. Daly, Ph.D.. "Final Report for Grant No. DE-FG02-97ER62492 "Engineering Deinococcus radiodurans for Metal Remediation in Radioactive Mixed Waste Sites". United States. doi:10.2172/837824.
Michael J. Daly, Ph.D.. 2005. ""Final Report for Grant No. DE-FG02-97ER62492 "Engineering Deinococcus radiodurans for Metal Remediation in Radioactive Mixed Waste Sites"". United States. doi:10.2172/837824. https://www.osti.gov/servlets/purl/837824.
@article{osti_837824,
title = {"Final Report for Grant No. DE-FG02-97ER62492 "Engineering Deinococcus radiodurans for Metal Remediation in Radioactive Mixed Waste Sites"},
author = {Michael J. Daly, Ph.D.},
abstractNote = {The groundwater and sediments of numerous U. S. Department of Energy (DOE) field sites are contaminated with mixtures of heavy metals (e.g., Hg, Cr, Pd) and radionuclides (e.g., U, Tc), as well as the fuel hydrocarbons benzene, toluene, ethylbenzene and xylenes (BTEX); chlorinated hydrocarbons, such as trichloroethylene (TCE); and polychlorinated biphenyls (PCBs). The remediation of such mixed wastes constitutes an immediate and complex waste management challenge for DOE, particularly in light of the costliness and limited efficacy of current physical and chemical strategies for treating mixed wastes. In situ bioremediation via natural microbial processes (e.g., metal reduction) remains a potent, potentially cost-effective approach to the reductive immobilization or detoxification of environmental contaminants. Seventy million cubic meters of soil and three trillion liters of groundwater have been contaminated by leaking radioactive waste generated in the United States during the Cold War. A cleanup technology is being developed based on the extremely radiation resistant bacterium Deinococcus radiodurans. Our recent isolation and characterization of D. radiodurans from a variety of DOE environments, including highly radioactive sediments beneath one of the leaking tanks (SX-108) at the Hanford Site in south-central Washington state, underscores the potential for this species to survive in such extreme environments. Research aimed at developing D. radiodurans for metal remediation in radioactive waste sites was started by this group in September 1997 with support from DOE NABIR grant DE-FG02-97ER62492. Our grant was renewed for the period 2000-2003, which includes work on the thermophilic radiation resistant bacterium Deinococcus geothermalis. Work funded by the existing grant contributed to 18 papers in the period 1997-2004 on the fundamental biology of D. radiodurans and its design for bioremediation of radioactive waste environments. Our progress since September 2000 closely matches the Aims proposed in our second NABIR application and is summarized as follows. We have further refined expression vectors for D. radiodurans and successfully tested engineered strains in natural DOE sediment and groundwater samples. Further, we have shown that D. geothermalis is transformable with plasmids and integration vectors designed for D. radiodurans. This was demonstrated by engineering Hg(II)-resistant D. geothermalis strains capable of reducing Hg(II) at elevated temperatures and under chronic irradiation. Additionally, we showed that D. geothermalis, like D. radiodurans, is naturally capable of reducing U(VI), Cr(VI), and Fe(III). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55 C, of which there are many examples. Our annotation of the D. radiodurans genome has been an important guide throughout this project period and continues to be a source of inspiration in the development of new genetic technologies dedicated to this bacterium. For example, our genome analyses have enabled us to achieve engineering goals that were unattainable in our first NABIR project (1997-2000), where uncertainties relating to its metabolic configuration prevented efforts to expand its metabolic capabilities. As just one example, we showed that D. radiodurans has a functioning tricarboxylic acid (TCA) cycle glyoxylate bypass which could be integrated with toluene oxidation. And, we successfully engineered D. radiodurans to derive carbon and energy from complete toluene mineralization and showed that toluene oxidation can be coupled to cellular biosynthesis, survival, as well as its native and engineered metal reducing capabilities. We have also constructed a whole genome microarray for D. radiodurans covering {approx}94% of its predicted genes and have successfully used the array to examine the response of cells to radiation and other DOE relevant conditions. Similarly, we have used high throughput proteomic approaches to examine how D. radiodurans responds to a variety of stress conditions. These studies have validated our annotation and are facilitating analysis of its metabolism, resistance, and metal reduction pathways.},
doi = {10.2172/837824},
journal = {},
number = ,
volume = ,
place = {United States},
year = 2005,
month = 3
}

Technical Report:

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  • Extremophiles are nearly always defined with singular characteristics that allow existence within a singular extreme environment. The bacterium Deinococcus radiodurans qualifies as a polyextremeophile, showing remarkable resistance to a range of damage caused by ionizing radiation, dessication, ultraviolet radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is most famous for its extreme resistance to ionizing radiation; it not only can grow continuously in the presence of chronic radiation (6,000 rad per hour), but it can survive acute exposures to gamma radiation that exceed 1,500,000 rads without lethality or induced mutation. These characteristics were the impetus for sequencing its genome. Wemore » completed an extensive comparative sequence analysis of the Deinococcus radiodurans (strain R1) genome. Deinococcus is the first representative with a completely sequenced genome from a bacterial branch of extremophiles - the Thermus/Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, support that it is a very ancient branch localized in the vicinity of the bacterial tree root. Distinctive features of the Deinoccoccus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to a collection of Clusters of Orthologous Groups of proteins (COGs). Analysis of paralogs in Deinococcus has revealed some unique protein families. In addition, specific expansions of several protein families including phosphatases, proteases, acyl transferases and MutT pyrophosphohydrolases, were detected. Genes that potentially affect DNA repair and recombination were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes, and are not present in other bacteria. For example, three proteins homologous to plant desiccation-resistance proteins were identified and these are particularly interesting because of the positive correlation between desiccation- and radiation-resistance. Further, the D. radiodurans genome is very rich in repetitive sequences, namely IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism. The genetic mechanisms underlying the extreme radiation resistance of this organism are now being characterized experimentally using a whole genome microarray.« less
  • Bacteria belonging to the family Deinococcaceae are some of the most ionizing radiation (IR) resistant organisms yet discovered. Deinococcus radiodurans is obligate aerobic, capable of growth under chronic IR (60 Gy/hour) and relatively resistant to many DNA damaging conditions including exposure to desiccation, ultraviolet radiation and hydrogen peroxide. The genes and cellular pathways underlying the survival strategies of D. radiodurans have been under investigation for fifty years. In the last decade, D. radiodurans was subjected to whole-genome sequencing, annotation and comparative analysis, whole-transcriptome and whole-proteome analyses, and numerous DNA repair studies. Collectively, published reports support that the key to survivalmore » of D. radiodurans resides in its ability to repair DNA, but the mechanisms responsible remain poorly defined. Unexpectedly, many novel genes implicated in recovery from IR by transcriptome and proteome profiling have had little effect on survival when disrupted, and there is reason to ask if something is missing from classical models of radiation resistance. The prevailing dogma of radiation toxicity has been that the cytotoxic and mutagenic effects of radiation are principally the result of DNA damage that occurs during IR. However, in light of available whole genome sequences, one broad observation that is difficult to reconcile with this view is that many organisms that encode a compliment of DNA repair and protection functions are killed at radiation doses that cause little DNA damage. This indicates that there are cellular targets involved in recovery that are more vulnerable to IR damage than DNA. It has been reported that D. radiodurans and other resistant organisms accumulate very high intracellular concentrations of Mn(II), and restricting the amount of Mn(II) during recovery from IR substantially reduced survival of D. radiodurans. At high intracellular concentrations, Mn(II) is known to act as a true catalyst of the dismutase of superoxde (O2?-), with Mn cycling between the divalent and trivalent states. Superoxide is generated during water radiolysis, particularly in the presence of iron redox-cycling processes, and has been implicated in damaging [4Fe-4S] cluster-containing proteins, with the release of bound Fe(II). Thus, it is possible that Mn(II) accumulation acts as keystone among antioxidant defenses which limits protein damage during both irradiation and recovery, with the result that DNA repair and other enzymic systems involved in recovery function with greater efficiency in D. radiodurans than other organisms.« less
  • 'A 1992 survey of DOE waste sites indicates that about 32% of soils and 45% of groundwaters at these sites contain radionuclides and metals plus an organic toxin class. The most commonly reported combinations of these hazardous compounds being radionuclides and metals (e.g., U, Pu, Cs, Pb, Cr, As) plus chlorinated hydrocarbons (e.g., trichloroethylene), fuel hydrocarbons (e.g., toluene), or polychlorinated biphenyls (e.g., Arochlor 1248). These wastes are some of the most hazardous pollutants and pose an increasing risk to human health as they leach into the environment. The objective of this research is to develop novel organisms, that are highlymore » resistant to radiation and the toxic effects of metals and radionuclides, for in-situ bioremediation of organic toxins. Few organisms exist that are able to remediate such environmental organic pollutants, and among those that can, the bacteria belonging to the genus Pseudomonas are the most characterized. Unfortunately, these bacteria are very radiation sensitive. For example, Pseudomonas spp. is even more sensitive than Escherichia coli and, thus, is not suitable as a bioremediation host in environments subjected to radiation. By contrast, D. radiodurans, a natural soil bacterium, is the most radiation resistant organism yet discovered; it is several thousand times more resistant to ionizing radiation than Pseudomonas. The sophisticated gene transfer and expression systems the authors have developed for D. radiodurans over the last eight years make this organism an ideal candidate for high-level expression of genes that degrade organic toxins, in radioactive environments. The authors ultimate aim is to develop organisms and approaches that will be useful for remediating the large variety of toxic organic compounds found in DOE waste sites that are too radioactive to support other bioremediation organisms. This report summarizes work after the first 6 months of a 3-year project.'« less
  • Seventy million cubic meters of ground and three trillion liters of groundwater have been contaminated by leaking radioactive waste generated in the United States during the Cold War. A cleanup technology is being developed based on the extremely radiation resistant bacterium Deinococcus radiodurans that is being engineered to express bioremediating functions. Research aimed at developing D. radiodurans for organic toxin degradation in highly radioactive waste sites containing radionuclides, heavy metals, and toxic organic compounds was started by this group.Work funded by the existing grant has already contributed to eleven papers on the fundamental biology of D. radiodurans and its designmore » for bioremediation of highly radioactive waste environments« less
  • Immense volumes of radioactive waste, generated from nuclear weapons production during the Cold War, were disposed directly to the ground. The current expense of remediating these polluted sites is driving the development of alternative remediation strategies using microorganisms. The bacterium Deinococcus radiodurans is the most radiation resistant organism known and can grow in highly irradiating (>60 Gray/h) environments (1). Numerous microorganisms (e.g., Pseudomonas sp.) have been described, and studied in detail, for their ability to transform and degrade a variety of organic pollutants (e.g., toluene), present at many radioactive DOE waste sites. Detoxification of the organic toxins at these sitesmore » is an important goal in remediating or stabilizing contaminated sites as well as preventing their further dissemination. The aim of this project is to engineer strains of D. radiodurans that are capable of degrading organic/aromatic hydrocarbons present in radioactive mixed waste sites--sites that contain mixtures of toxic organic compounds, radionuclides and heavy metals. Conventional bioremediating organisms are unable to survive at many of these sites because of their sensitivity to radiation. Generally, microorganisms are sensitive to the damaging effects of ionizing radiation, and most of the bacteria currently being studied as candidates for bioremediation are no exception. For example, Pseudomonas sp. is very sensitive to radiation (more sensitive than E. coli) and is not suited to remediate radioactive wastes. Therefore, radiation resistant microorganisms that can remediate toxic organic compounds need to be found in nature or engineered in the laboratory to address this problem.« less