Responses of Mn\u3csup\u3e2+\u3c/sup\u3e Speciation in \u3cem\u3eDeinococcus radiodurans\u3c/em\u3e and \u3cem\u3eEscherichia coli\u3c/em\u3e to γ-Radiation by Advanced Paramagnetic Resonance Methods

The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of γ-rays (12,000 Gy), 20 times greater than Escherichia coli, is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) “antioxidant” Mn2+–metabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)–scavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn2+ speciation in D. radiodurans and E. coli cells and their responses to 10 kGy γ-irradiation. The Mn2+ of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn2+ of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn2+ speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn2+ complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn2+ speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation-induced ROS because it lacks an adequate supply of LMW Mn antioxidants

Microbial cells can cooperate to resist high-level chronic ionizing radiation

Understanding chronic ionizing radiation (CIR) effects is of utmost importance to protecting human health and the environment. Diverse bacteria and fungi inhabiting extremely radioactive waste and disaster sites (e.g. Hanford, Chernobyl, Fukushima) represent new targets of CIR research. We show that many microorganisms can grow under intense gamma-CIR dose rates of 13–126 Gy/h, with fungi identified as a particularly CIR-resistant group of eukaryotes: among 145 phylogenetically diverse strains tested, 78 grew under 36 Gy/h. Importantly, we demonstrate that CIR resistance can depend on cell concentration and that certain resistant microbial cells protect their neighbors (not only conspecifics, but even radiosensitive species from a different phylum), from high-level CIR. We apply a mechanistically-motivated mathematical model of CIR effects, based on accumulation/removal kinetics of reactive oxygen species (ROS) and antioxidants, in bacteria (3 Escherichia coli strains and Deinococcus radiodurans) and in fungi (Candida parapsilosis, Kazachstania exigua, Pichia kudriavzevii, Rhodotorula lysinophila, Saccharomyces cerevisiae, and Trichosporon mucoides). We also show that correlations between responses to CIR and acute ionizing radiation (AIR) among studied microorganisms are weak. For example, in D. radiodurans, the best molecular correlate for CIR resistance is the antioxidant enzyme catalase, which is dispensable for AIR resistance; and numerous CIR-resistant fungi are not AIR-resistant. Our experimental findings and quantitative modeling thus demonstrate the importance of investigating CIR responses directly, rather than extrapolating from AIR. Protection of radiosensitive cell-types by radioresistant ones under high-level CIR is a potentially important new tool for bioremediation of radioactive sites and development of CIR-resistant microbiota as radioprotectors

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

Across the tree of life, radiation resistance is governed by antioxidant Mn2+, gauged by paramagnetic resonance

Despite concerted functional genomic efforts to understand the complex phenotype of ionizing radiation (IR) resistance, a genome sequence cannot predict whether a cell is IR-resistant or not. Instead, we report that absorption-display electron paramagnetic resonance (EPR) spectroscopy of nonirradiated cells is highly diagnostic of IR survival and repair efficiency of DNA double-strand breaks (DSBs) caused by exposure to gamma radiation across archaea, bacteria, and eukaryotes, including fungi and human cells. IR-resistant cells, which are efficient at DSB repair, contain a high cellular content of manganous ions (Mn2+) in high-symmetry (H) antioxidant complexes with small metabolites (e.g., orthophosphate, peptides), which exhibit narrow EPR signals (small zero-field splitting). In contrast, Mn2+ ions in IR-sensitive cells, which are inefficient at DSB repair, exist largely as low-symmetry (L) complexes with substantially broadened spectra seen with enzymes and strongly chelating ligands. The fraction of cellular Mn2+ present as H-complexes (H-Mn2+), as measured by EPR of live, nonirradiated Mn-replete cells, is now the strongest known gauge of biological IR resistance between and within organisms representing all three domains of life: Antioxidant H-Mn2+ complexes, not antioxidant enzymes (e.g., Mn superoxide dismutase), govern IR survival. As the pool of intracellular metabolites needed to form H-Mn2+ complexes depends on the nutritional status of the cell, we conclude that IR resistance is predominantly a metabolic phenomenon. In a cross-kingdom analysis, the vast differences in taxonomic classification, genome size, and radioresistance between cell types studied here support that IR resistance is not controlled by the repertoire of DNA repair and antioxidant enzymes

Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance

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

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

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

Site-specific strand breaks in RNA produced by (125)I radiodecay

Decay of (125)I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of (125)I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3′-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when (125)I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that (125)I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules

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

High-quality genome sequence of the radioresistant bacterium Deinococcus ficus KS 0460

The genetic platforms of Deinococcus species remain the only systems in which massive ionizing radiation (IR)-induced genome damage can be investigated in vivo at exposures commensurate with cellular survival. We report the whole genome sequence of the extremely IR-resistant rod-shaped bacterium Deinococcus ficus KS 0460 and its phenotypic characterization. Deinococcus ficus KS 0460 has been studied since 1987, first under the name Deinobacter grandis, then Deinococcus grandis. The D. ficus KS 0460 genome consists of a 4.019 Mbp sequence (69.7% GC content and 3894 predicted genes) divided into six genome partitions, five of which are confirmed to be circular. Circularity was determined manually by mate pair linkage. Approximately 76% of the predicted proteins contained identifiable Pfam domains and 72% were assigned to COGs. Of all D. ficus KS 0460 proteins, 79% and 70% had homologues in Deinococcus radiodurans ATCC BAA-816 and Deinococcus geothermalis DSM 11300, respectively. The most striking differences between D. ficus KS 0460 and D. radiodurans BAA-816 identified by the comparison of the KEGG pathways were as follows: (i) D. ficus lacks nine enzymes of purine degradation present in D. radiodurans, and (ii) D. ficus contains eight enzymes involved in nitrogen metabolism, including nitrate and nitrite reductases, that D. radiodurans lacks. Moreover, genes previously considered to be important to IR resistance are missing in D. ficus KS 0460, namely, for the Mn-transporter nramp, and proteins DdrF, DdrJ and DdrK, all of which are also missing in Deinococcus deserti. Otherwise, D. ficus KS 0460 exemplifies the Deinococcus lineage