153. Functional Analysis of Deinococcus radiodurans Genomes by Targeted Mutagenesis 

Kwong-Kwok Wong, William B. Chrisler, Lye Meng Markillie, and Richard D. Smith 
Pacific Northwest National Laboratory, Molecular Biosciences, MS P7-56, P.O. Box 999, Richland, WA 99352 
kk.wong@pnl.gov 

D. radiodurans, previously known as Micrococcus radiodurans, strains R1, has extreme resistance to genotoxic chemicals, oxidative damage, high levels of ionizing and UV radiation, and desiccation. The ability to survive such extreme environments is attributed in part to a unique DNA repair system in combination with its chromosome copy number and structure, as well as factors affecting the survival of other cellular components. There is evidence suggesting that the carotenoids which cause red pigmentation in D. radiodurans may act as free radical scavengers, thus increasing resistance to DNA damage by hydroxyl radicals. High levels of two oxygen toxicity defense enzymes, superoxide dismutase and catalase, are also found in D. radiodurans. In addition, the Deinococcal outer membrane lipids are complex and distinct from those found in the rest of the bacterial world and it has been suggested that they, together with the plasma membrane, may also be involved in stress resistance. However, the genetic basis for these stress resistance is still not clear. With the genomic sequence information of D. radiodurans R1, we have developed a simple and general targeted mutagenesis method to perform a genome-wide analysis of putative genes involved in the stress resistance. We have generated mutations in katA (catalase) and sodA (superoxide dismutase). Both katA and sodA mutants are shown to be required for the extreme ionizing radiation resistance. Several other mutations have been generated and are being analyzed for their roles in stress resistance.