THE ROLE OF IRON IN Deinococcus radiodurans ENGINEERED FOR GROWTH ON TOLUENE AND THE ROLE OF MANGANESE IN THE EXTREME RADIATION RESISTANCE PHENOTYPE

Toluene and other fuel hydrocarbons are commonly found in association with radionuclides at numerous Department of Energy (DOE) sites, frequently occurring together with Cr(VI) and other heavy metals. In this study, the extremely radiation resistant bacterium Deinococcus radiodurans was engineered for complete toluene mineralization by cloned expression of tod and xyl genes of Pseudomonas putida. The recombinant Tod/Xyl strain showed significant incorporation of carbon from the toluene aromatic ring into cellular macromolecules and carbon dioxide, in the absence or presence of chronic radiation. We have shown that intracellular iron concentrations in wild-type D. radiodurans in minimal medium are exceptionally low and not sufficient to support growth on toluene using Fe-dependent oxygenases cloned from P. putida. Introducing the fur mutation into D. radiodurans increased intracellular Fe levels, and imparted on the engineered strain the ability to grow on meta-toluate as the sole carbon and energy source. The organism's native Cr(VI) reduction capabilities were facilitated by toluene when present as the sole carbon and energy source in natural sediment analogues of DOE contaminated environments. The engineered bacteria were able to oxidize toluene under both minimal and complex nutrient conditions, which is important since both conditions have environmental equivalents in the context of bioremediation processes. As such, the Tod/Xyl strain is providing a model for understanding the role of Fe and reduction of metals coupled to organic contaminant oxidation in aerobic radionuclide contaminated sediments. We have shown that D. radiodurans contains high intracellular manganese levels, and that Mn restriction sensitizes cells to irradiation. We propose that the unusually high Mn/Fe ratio of D. radiodurans facilitates survival by quenching oxidative stress during recovery.