Forty-one ionizing radiation-sensitive strains of Deinococcus radiodurans were evaluated for their ability to survive 6 weeks of desiccation. All exhibited a substantial loss of viability upon rehydration compared with wild-type D. radiodurans. Examination of chromosomal DNA from desiccated cultures revealed a time-dependent increase in DNA damage, as measured by an increase in DNA double-strand breaks. The evidence presented suggests that D. radiodurans' ionizing radiation resistance is incidental, a consequence of this organism's adaptation to a common physiological stress, dehydration.The Deinococcaceae are a small family of non-spore-forming bacteria which exhibit a remarkable capacity to resist the lethal effects of ionizing radiation (10,11,18). Well-aerated, exponential-phase cultures of members of this family will survive 5,000 Gy of gamma radiation without loss of viability (14), and survivors are routinely recovered from cultures exposed to as much as 20 kGy (1, 7). Of the five species that make up the Deinococcaceae, Deinococcus radiodurans has been most extensively studied, and it has been determined that the radioresistance of this species is a direct result of its ability to efficiently repair the DNA damage generated during irradiation (10,11,18). In other words, the extreme radioresistance of D. radiodurans-and presumably the other deinococci-appears to be the result of an evolutionary process that selected for organisms that could tolerate massive DNA damage. The reasons for D. radiodurans' ionizing radiation resistance are obscure, however. The deinococci's radioresistance cannot be an adaptation (i.e., an evolutionary modification of a character under selection) to ionizing radiation, because there is no selective advantage to being ionizing radiation resistant in the natural world. There are no terrestrial environments that generate such a high flux of ionizing radiation (20). It must therefore be assumed that the deinococci's radioresistance is an incidental use of the cell's DNA repair capability. In this study, we ask why the DNA repair capabilities of D. radiodurans evolved, and we provide evidence suggesting that they were built by selection for desiccation resistance. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. All D. radiodurans strains were grown at 30ЊC in TGY broth (0.5% tryptone, 0.3% yeast extract, 0.1% glucose) or on TGY agar (TGY broth with 1.5% agar).Quantifying desiccation resistance. Cells from an exponential-phase culture of each strain examined were collected by centrifugation, washed in 4 volumes of 10 mM MgSO 4 , and resuspended in an equal volume of 10 mM MgSO 4 . A 100-l aliquot of this suspension was spotted on a sterile glass coverslip, placed inside a sterile petri dish, and dried at 25ЊC in a desiccator over anhydrous CaSO 4 containing a visual indicator. The desiccators were sealed, and the dried cultures were stored undisturbed at 25ЊC for 6 weeks. Relative humidity within the de...

Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of either locus results in a partial loss of resistance to ionizing radiation. The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant. In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation. The irrB and irrI mutations also reduce D. radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds. Subclones of each gene have been isolated, and the loci have been mapped relative to each other. The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes.

Natural transformation was used to help define a collection of ionizing radiation-sensitive strains of Deinococcus radiodurans. Three putative rec mutations were identified, as were three pol alleles. Forty of the ionizing radiation-sensitive strains were placed into 16 linkage groups, and evidence obtained indicates that each linkage group consists of a cluster of mutations not more than 1,000 bp apart. In addition, a new class of D. radiodurans mutant was described that, although radioresistant, appears to recover from ionizing radiation-induced DNA damage slowly relative to other strains of D. radiodurans.Deinococcus radiodurans is the most-studied member of the family Deinococcaceae, a bacterial family characterized by extreme resistance to ionizing radiation (11,13,17). Well-aerated, exponential-phase cultures of D. radiodurans survive 5,000 Gy of ␥ radiation without loss of viability (16), and there are reports of other deinococcal strains surviving as much as 50 kGy (3) of ␥ radiation. Despite this remarkable property and the potential advantages such an organism offers to those studying ionizing radiation-induced DNA damage and its repair, the mechanisms responsible for the extreme radioresistance of the deinococci are poorly understood.While the capacity to survive the massive DNA damage inflicted by extreme doses of ionizing radiation certainly suggests that the deinococci have evolved sophisticated DNA repair systems to deal with that damage, there is little direct evidence to support this conclusion. Only four deinococcal proteins have been associated with ionizing radiation resistance. They are the rec and pol gene products, homologs of Escherichia coli RecA (8) and DNA polymerase I (9), respectively, and the irrB and irrI gene products, whose biochemical functions have yet to be determined (20).During previous studies of D. radiodurans, we isolated 49 putative ionizing radiation-sensitive (IRS) strains with the intention of using these strains to define the enzymatic components involved in deinococcal ionizing radiation resistance. We further characterize this collection of strains here by demonstrating (i) that the mutations responsible for the IRS phenotype fall into 16 different linkage groups, (ii) that all but three of the IRS strains are recombination proficient as evidenced by their ability to undergo natural transformation, (iii) that three of the IRS strains carry mutations in the D. radiodurans pol locus, and (iv) that five of the strains in this collection recover from ␥ radiation-induced DNA damage at a significantly slower rate than does their parent, D. radiodurans 302. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. All D. radiodurans strains were grown at 30ЊC in TGY broth (0.5% tryptone, 0.3% yeast extract, 0.1% glucose) or on TGY agar (1.5% agar). Plasmids were routinely propagated in E. coli DH5␣-MCR. Plasmids were isolated by alkaline extraction (4).N-Methyl-N-nitro-N-nitrosoguanidine mutagenesis a...

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