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1.  Effects of Mn and Fe Levels on Bacillus subtilis Spore Resistance and Effects of Mn2+, Other Divalent Cations, Orthophosphate, and Dipicolinic Acid on Protein Resistance to Ionizing Radiation ▿  
Spores of Bacillus subtilis strains with (wild type) or without (α−β−) most DNA-binding α/β-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl2 concentrations of 0.3 μM to 1 mM. These haploid spores had Mn levels that varied up to 180-fold and Mn/Fe ratios that varied up to 300-fold. However, the resistance of these spores to desiccation, wet heat, dry heat, and in particular ionizing radiation was unaffected by their level of Mn or their Mn/Fe ratio; this was also the case for wild-type spore resistance to hydrogen peroxide (H2O2). However, α−β− spores were more sensitive to H2O2 when they had high Mn levels and a high Mn/Fe ratio. These results suggest that Mn levels alone are not essential for wild-type bacterial spores' extreme resistance properties, in particular ionizing radiation, although high Mn levels sensitize α−β− spores to H2O2, probably by repressing expression of the auxiliary DNA-protective protein MrgA. Notably, Mn2+ complexed with the abundant spore molecule dipicolinic acid (DPA) with or without inorganic phosphate was very effective at protecting a restriction enzyme against ionizing radiation in vitro, and Ca2+ complexed with DPA and phosphate was also very effective in this regard. These latter data suggest that protein protection in spores against treatments such as ionizing radiation that generate reactive oxygen species may be due in part to the spores' high levels of DPA conjugated to divalent metal ions, predominantly Ca2+, much like high levels of Mn2+ complexed with small molecules protect the bacterium Deinococcus radiodurans against ionizing radiation.
doi:10.1128/AEM.01965-10
PMCID: PMC3019732  PMID: 21057011
2.  Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans 
PLoS ONE  2010;5(9):e12570.
For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn2+ and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn2+-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.
doi:10.1371/journal.pone.0012570
PMCID: PMC2933237  PMID: 20838443
3.  Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance  
PLoS Biology  2007;5(4):e92.
In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.
Author Summary
One original goal of radiobiology was to explain why cells are so sensitive to ionizing radiation (IR). Early studies in bacteria incriminated DNA as the principal radiosensitive target, an assertion that remains central to modern radiation toxicity models. More recently, the emphasis has shifted to understanding why bacteria such as Deinococcus radiodurans are extremely resistant to IR, by focusing on DNA repair systems expressed during recovery from high doses of IR. Unfortunately, as key features of DNA-centric hypotheses of extreme resistance have grown weaker, the study of alternative cellular targets has lagged far behind, mostly because of their relative biological complexity. Recent studies have shown that extreme levels of bacterial IR resistance correlate with high intracellular Mn(II) concentrations, and resistant and sensitive bacteria are equally susceptible to IR-induced DNA damage. The current work establishes a mechanistic link between Mn(II) and protection of proteins from radiation damage. In contrast to resistant bacteria, naturally sensitive bacteria are highly susceptible to IR-induced protein oxidation. We propose that sensitive bacteria sustain lethal levels of protein damage at radiation doses that elicit relatively little DNA damage, and that extreme resistance in bacteria is dependent on protein protection.
A high intracellular concentration of manganese inDeinococcus radiodurans protects proteins, but not DNA, from ionizing radiation-induced oxidative damage. Protein protection may be critical to the known radiation resistance of these bacteria.
doi:10.1371/journal.pbio.0050092
PMCID: PMC1828145  PMID: 17373858
4.  Transcriptome Analysis Applied to Survival of Shewanella oneidensis MR-1 Exposed to Ionizing Radiation 
Journal of Bacteriology  2006;188(3):1199-1204.
The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of DNA repair systems and 40 Gy IR induces less than one double-strand break in its genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this organism's great sensitivity to IR.
doi:10.1128/JB.188.3.1199-1204.2006
PMCID: PMC1347324  PMID: 16428429
5.  Comparative genomics of Thermus thermophilus and Deinococcus radiodurans: divergent routes of adaptation to thermophily and radiation resistance 
Background
Thermus thermophilus and Deinococcus radiodurans belong to a distinct bacterial clade but have remarkably different phenotypes. T. thermophilus is a thermophile, which is relatively sensitive to ionizing radiation and desiccation, whereas D. radiodurans is a mesophile, which is highly radiation- and desiccation-resistant. Here we present an in-depth comparison of the genomes of these two related but differently adapted bacteria.
Results
By reconstructing the evolution of Thermus and Deinococcus after the divergence from their common ancestor, we demonstrate a high level of post-divergence gene flux in both lineages. Various aspects of the adaptation to high temperature in Thermus can be attributed to horizontal gene transfer from archaea and thermophilic bacteria; many of the horizontally transferred genes are located on the single megaplasmid of Thermus. In addition, the Thermus lineage has lost a set of genes that are still present in Deinococcus and many other mesophilic bacteria but are not common among thermophiles. By contrast, Deinococcus seems to have acquired numerous genes related to stress response systems from various bacteria. A comparison of the distribution of orthologous genes among the four partitions of the Deinococcus genome and the two partitions of the Thermus genome reveals homology between the Thermus megaplasmid (pTT27) and Deinococcus megaplasmid (DR177).
Conclusion
After the radiation from their common ancestor, the Thermus and Deinococcus lineages have taken divergent paths toward their distinct lifestyles. In addition to extensive gene loss, Thermus seems to have acquired numerous genes from thermophiles, which likely was the decisive contribution to its thermophilic adaptation. By contrast, Deinococcus lost few genes but seems to have acquired many bacterial genes that apparently enhanced its ability to survive different kinds of environmental stresses. Notwithstanding the accumulation of horizontally transferred genes, we also show that the single megaplasmid of Thermus and the DR177 megaplasmid of Deinococcus are homologous and probably were inherited from the common ancestor of these bacteria.
doi:10.1186/1471-2148-5-57
PMCID: PMC1274311  PMID: 16242020
6.  Geomicrobiology of High-Level Nuclear Waste-Contaminated Vadose Sediments at the Hanford Site, Washington State 
Sediments from a high-level nuclear waste plume were collected as part of investigations to evaluate the potential fate and migration of contaminants in the subsurface. The plume originated from a leak that occurred in 1962 from a waste tank consisting of high concentrations of alkali, nitrate, aluminate, Cr(VI), 137Cs, and 99Tc. Investigations were initiated to determine the distribution of viable microorganisms in the vadose sediment samples, probe the phylogeny of cultivated and uncultivated members, and evaluate the ability of the cultivated organisms to survive acute doses of ionizing radiation. The populations of viable aerobic heterotrophic bacteria were generally low, from below detection to ∼104 CFU g−1, but viable microorganisms were recovered from 11 of 16 samples, including several of the most radioactive ones (e.g., >10 μCi of 137Cs/g). The isolates from the contaminated sediments and clone libraries from sediment DNA extracts were dominated by members related to known gram-positive bacteria. Gram-positive bacteria most closely related to Arthrobacter species were the most common isolates among all samples, but other phyla high in G+C content were also represented, including Rhodococcus and Nocardia. Two isolates from the second-most radioactive sample (>20 μCi of 137Cs g−1) were closely related to Deinococcus radiodurans and were able to survive acute doses of ionizing radiation approaching 20 kGy. Many of the gram-positive isolates were resistant to lower levels of gamma radiation. These results demonstrate that gram-positive bacteria, predominantly from phyla high in G+C content, are indigenous to Hanford vadose sediments and that some are effective at surviving the extreme physical and chemical stress associated with radioactive waste.
doi:10.1128/AEM.70.7.4230-4241.2004
PMCID: PMC444790  PMID: 15240306
7.  Engineering Deinococcus geothermalis for Bioremediation of High-Temperature Radioactive Waste Environments 
Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55°C.
doi:10.1128/AEM.69.8.4575-4582.2003
PMCID: PMC169113  PMID: 12902245
8.  RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization 
Journal of Bacteriology  2002;184(6):1649-1660.
The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecADr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecADr protein binds much better to duplex DNA than the RecAEc protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.
doi:10.1128/JB.184.6.1649-1660.2002
PMCID: PMC134872  PMID: 11872716
9.  Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics 
The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.
doi:10.1128/MMBR.65.1.44-79.2001
PMCID: PMC99018  PMID: 11238985
10.  Physiologic Determinants of Radiation Resistance in Deinococcus radiodurans 
Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium Deinococcus radiodurans is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of D. radiodurans and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that D. radiodurans can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of D. radiodurans and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete D. radiodurans genomic sequence support the hypothesis that there are several defects in D. radiodurans global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of D. radiodurans in nutritionally limiting radioactive environments.
PMCID: PMC110589  PMID: 10831446
11.  Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks 
PLoS ONE  2007;2(9):e955.
Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.
doi:10.1371/journal.pone.0000955
PMCID: PMC1978522  PMID: 17895995
12.  Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum 
Journal of Bacteriology  2001;183(16):4823-4838.
The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.
doi:10.1128/JB.183.16.4823-4838.2001
PMCID: PMC99537  PMID: 11466286

Results 1-12 (12)