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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...

During the first hour after a sublethal dose of ionizing radiation, 72 genes were upregulated threefold or higher in D. radiodurans R1. Thirty-three of these loci were also among a set of 73 genes expressed in R1 cultures recovering from desiccation. The five transcripts most highly induced in response to each stress are the same and encode proteins of unknown function. The genes (ddrA, ddrB, ddrC, ddrD, and pprA) corresponding to these transcripts were deleted, both alone and in all possible two-way combinations. Characterization of the mutant strains defines three epistasis groups that reflect different cellular responses to ionizing radiation-induced damage. The ddrA and ddrB gene products have complementary activities and inactivating both loci generates a strain that is more sensitive to ionizing radiation than strains in which either single gene has been deleted. These proteins appear to mediate efficient RecA-independent processes connected to ionizing radiation resistance. The pprA gene product is not necessary for homologous recombination during natural transformation, but nevertheless may participate in a RecA-dependent process during recovery from radiation damage. These characterizations clearly demonstrate that novel mechanisms significantly contribute to the ionizing radiation resistance in D. radiodurans. DEINOCOCCUS radiodurans R1 is the type species tution of ionizing radiation-induced single-strand (Dean et al. 1969) and double-strand DNA breaks (Kitayama of a bacterial family distinguished by its ability to tolerate exposure to ionizing radiation (Battista et al. and Matsuyama 1971). We have made identifying the proteins that mediate ionizing radiation resistance a 1999); exponential phase cultures survive doses to 5 kGy without loss of viability. A 5-kGy dose causes massive priority in our efforts to better explain D. radiodurans's extreme radioresistance. DNA damage, cleaving the genome of every D. radiodurans cell into multiple, subgenomic fragments (Bat-In this report we have described the genomic expression profile of D. radiodurans R1 cultures as they recover tista et al. 1999). For most species, this level of DNA damage is lethal, but D. radiodurans has the capacity to from a sublethal dose of ionizing radiation and compare that profile with R1 cultures recovering from desiccareform its genome from these fragments in what appears to be an error-free process. The biochemical details of tion (Mattimore and Battista 1996) to define the overlap in the D. radiodurans response to these stresses. D. radiodurans's ionizing radiation resistance are poorly understood, but it is clear that proteins needed for cell Mattimore and Battista (1996) established a link between the desiccation resistance and the radiotolerance survival are synthesized in cultures exposed to ionizing radiation. Irradiated cultures cannot recover in the presof D. radiodurans by demonstrating that a collection of ionizing radiation-sensitive strains were also sensitive to ence of chloramphenicol; this antibiotic p...

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Bacteria of the genus Deinococcus exhibit an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents-particularly the effects of ionizing radiation. These bacteria are the most DNA damage-tolerant organisms ever identified. Relatively little is known about the biochemical basis for this phenomenon; however, available evidence indicates that efficient repair of DNA damage is, in large part, responsible for the deinococci's radioresistance. Obviously, an explanation of the deinococci's DNA damage tolerance cannot be developed solely on the basis of the DNA repair strategies of more radiosensitive organisms. The deinococci's capacity to survive DNA damage suggests that (a) they employ repair mechanisms that are fundamentally different from other prokaryotes, or that (b) they have the ability to potentiate the effectiveness of the conventional complement of DNA repair proteins. An argument is made for the latter alternative.