New technology may reveal mechanisms of radiation resistance in Deinococcus radiodurans

Advances in DNA sequencing technology in the past decade have resulted in rapid production of DNA data including more than 70 complete prokaryotic genomes and several eukaryotic genomes currently available. This accumulation of sequence data has provided opportunities for development of proteomics. Proteomic analyses focus on composition of the protein mixture extracted from a cell culture and its changes in response to environmental disturbances or during the cell cycle. A standard scenario is as follows: First, extract the complex protein mixture from cell cultures growing under controlled conditions. Then run a two-dimensional PAGE to separate different proteins into a large number of often partly overlapping spots. Finally, identify the spots and assign them to known proteins or genes (e.g., ref. 1). This last step is facilitated by use of MS (e.g., ref. 2). Routine use of MS technology in proteomics was made possible by complete genome sequencing. A complete genomic sequence can be used to identify all putative proteins encoded in the genome. This limits the number of possible peptide fragments expected to be found in the proteins extracted from the cells (3) and, given sufficient accuracy of the MS measurements, the mass of a fragment can often be unambiguously associated with a specific protein. In this issue of PNAS, Lipton et al. (4) use an improved MS technology based on peptide accurate mass tags for proteomic analyses of the radiation-resistant bacterium Deinococcus radiodurans. The method uses Fourier transform ion cyclotron resonance (FTICR) experimentation instead of standard tandem MS, which provides greater sensitivity and accuracy, yielding a more comprehensive coverage of the proteome. Thus, the accurate FTICR measurements applied to D. radiodurans identified accurate mass tags for 61% of all putative D. radiodurans proteins—the most complete proteome coverage to date.

D. radiodurans' radioresistance makes it one of the leading candidates for bioremediation of radioactive waste sites.

D. radiodurans is exceptional in its capacity to withstand high doses of ionizing and UV radiation that are lethal to virtually all other living organisms. It is a nonpathogenic, red-pigmented bacterium. Although technically Gram-positive, D. radiodurans features plasma and outer membranes and a multilayer cell envelope reminiscent of Gram-negative bacteria (5). Its radioresistance makes it one of the leading candidates for bioremediation of radioactive waste sites that contain hazardous mixtures of radionuclides, heavy metals, and other toxic chemicals. Engineered strains of D. radiodurans are capable of transforming these mixtures to less hazardous ones by degrading some of the toxic components (6, 7). Understanding molecular mechanisms that allow cells to survive damaging effects of UV radiation is also of interest in medical research, especially in conjunction with ongoing environmental changes that allow more UV radiation to reach the Earth's surface (8).

Although the complete genomic DNA sequence of D. radiodurans is now known (9), the molecular mechanisms of its radiation resistance are not well understood. Radiation causes complex damage to various cell components, including nucleic acids, proteins, and membranes. The damage can be caused directly by the radiation or indirectly by free radicals generated by it. At the DNA level, UV radiation damage is limited mainly to DNA bases whereas ionizing radiation causes a wider variety of damage, including single- and double-strand breaks. The single-strand breaks, where one DNA strand is damaged but the other remains intact, can be repaired by base excision repair where the damaged part of the DNA is removed and synthesized de novo by using the undamaged strand as a template. In double-strand breaks, both DNA strands are broken at the same site, precluding excision repair. Different strategies can be used in protecting DNA from the effects of radiation. Rubrobacter radiotolerans, another radiation-resistant bacterium, apparently protects its DNA so the strand breaks do not occur (10). By contrast, D. radiodurans has been demonstrated to accumulate numerous double-strand DNA breaks during irradiation and invokes its extraordinary DNA repair facility to reconstruct its genome from the remaining fragments (11, 12). Similar repair mechanisms are also apparently involved in radioresistance of Pyrococcus furiosus (13) and possibly other hyperthermophilic archaea. Many bacteria including D. radiodurans possess multiple copies of their chromosomes and can recover from a limited number of double-strand DNA breaks by reassembling an intact chromosome from overlapping fragments via homologous recombination. However, multiple copies of chromosomes do not themselves confer radiation resistance. D. radiodurans can repair more than 100 double-strand DNA breaks without lethality whereas most other bacteria with multiple copies of chromosomes are unable to repair more than a few double-strand breaks (14). It is possible that D. radiodurans uses this type of repair in a different way than other organisms. Homologous recombination is promoted by the RecA protein, which plays an important role in the radiation resistance of D. radiodurans. When D. radiodurans RecA was replaced with a RecA gene from Shigella flexneri, the resulting D. radiodurans mutant lost its radiation resistance (11). The D. radiodurans RecA was originally not detected in undamaged cells, leading to speculation that it is actually toxic to the normal cells and is expressed only after DNA damage. However, precise accurate mass tag measurements detected RecA under normal growth conditions and confirmed its significant increase after irradiation (4).

In our laboratory, we identified predicted highly expressed (PHX) genes of D. radiodurans and compared them to other prokaryotes (15). With the exception of RecA, the DNA repair proteins of D. radiodurans are generally of low predicted expression levels and their assortment is not larger than that of Escherichia coli. The D. radiodurans RecA has a very high predicted expression level compared with RecA proteins in other prokaryotes, consistent with its possible enhanced role in D. radiodurans. On the other hand, in comparison with other prokaryotes, D. radiodurans stands out in its number of PHX chaperone, degradation, protease, and detoxification genes. Chaperones assist protein folding and repair misfolded proteins whereas degradation enzymes and proteases digest and recycle dysfunctional proteins and nucleic acids. Detoxification proteins remove toxic free radicals from the cell. We (15) speculated that chaperone, degradation, and detoxification proteins may play an important role in response to extensive damage caused by radiation. Degradation and export of damaged DNA after irradiation is an important part of the repair process (12) and the same probably applies to damaged proteins.

Complex damage to the cell, such as that caused by ionizing radiation, probably requires a complex cellular response, which may involve many different components. Among proteins whose production increases after irradiation are RecA, which is involved in DNA recombination, and also the translation elongation factor Tu required for protein synthesis (16). The concentration of catalase KatA also has been shown to increase after irradiation, and mutations in superoxide dismutase SodA diminish radioresistance of D. radiodurans (17). Catalases remove hydrogen peroxide whereas superoxide dismutases eliminate superoxide radicals from the cell. Both hydrogen peroxide and superoxide arise from irradiation of the cells. The pentose phosphate pathway converts glucose to precursors of deoxyribonucleotide triphosphates, which are used in DNA synthesis and repair. It has been proposed that an active pentose phosphate pathway is important in D. radiodurans radiation resistance (18). The protein Rsr has been shown to contribute to D. radiodurans resistance to UV radiation (although not ionizing radiation). Rsr is a homolog of ribonucleoprotein Ro previously found only in eukaryotes where it binds misfolded 5S rRNA molecules. Chen et al. (19) suggested that it could contribute to degradation and removal of damaged RNA molecules. Lipton et al. (4) detected Rsr in cell cultures growing under most conditions, but not after an extended period of starvation (4 weeks). D. radiodurans contains a high number of intergenic DNA repeats that could contribute to efficient recombination and enhance DNA repair after irradiation (9).

Besides its capacity to survive high levels of radiation, D. radiodurans can also withstand periods of desiccation. It has been suggested that radiation resistance evolved as a byproduct of desiccation resistance because highly radioactive environments do not naturally occur on Earth (5, 20). Desiccation inflicts similar complex damage to cellular components as high levels of radiation, and many desiccation-tolerant prokaryotes are also resistant to radiation (e.g., ref. 21). It is intriguing that D. radiodurans contains four proteins that are not found in other prokaryotes but whose homologs in plants are induced by desiccation (5). Among these proteins (labeled DR1372, DR0105, DR1172, and DRB0118) only DR1172 was detected under varying culture conditions, whereas DR0105 was detected only in stationary phase and exponential growth phase in defined medium (table 4 in ref. 4). DR1372 and DRB0118 were not detected under any of the culture conditions investigated, possibly indicating a highly specialized function for these proteins.

Although repair processes appear to play an important role in D. radiodurans' survival of radiation and desiccation damage, so far there is no evidence of repair mechanisms unique to D. radiodurans but not present in less resistant organisms. In fact, D. radiodurans DNA repair pathways appear to be less complex and diverse than those of E. coli or Bacillus subtilis. For example, the RecBCD recombinase, which is a major component of recombination systems in most free-living organisms, is not found in the D. radiodurans genome (5). However, the function of a large number of D. radiodurans proteins has yet to be determined, and some of these may turn out to be the missing pieces of the puzzle. Notably, D. radiodurans contains an unusually high number of uncharacterized hypothetical proteins whose codon usage is consistent with high expression levels (15). As an example of how their data could be used in characterizing hypothetical proteins, Lipton et al. (4) predict a functional classification for the protein DR0070. It has weak similarity to an alkaline protease of B. subtilis, which itself would not be sufficient to confidently classify the protein. However, the DR0070 protein was detected only in response to alkaline shock and not under any other culture condition. Alkaline proteases are most stable and have optimal activity at high pH, and therefore this expression pattern is consistent with the classification as an alkaline protease.

In the work of Lipton et al. (4), we are presented with a comprehensive set of data on the D. radiodurans proteome, but this is only the first step. Thorough analysis of these data combined with additional experiments may reveal the mechanisms that help living cells survive high levels of radiation.