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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

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