Effects of Desiccation and Freezing on Microbial Ionizing Radiation Survivability: Considerations for Mars Sample Return.

Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.

Small-Molecule Mn Antioxidants in Caenorhabditis elegans and Deinococcus radiodurans Supplant MnSOD Enzymes during Aging and Irradiation.

Denham Harman's oxidative damage theory identifies superoxide (O2•-) radicals as central agents of aging and radiation injury, with Mn2+-dependent superoxide dismutase (MnSOD) as the principal O2•--scavenger. However, in the radiation-resistant nematode Caenorhabditis elegans, the mitochondrial antioxidant enzyme MnSOD is dispensable for longevity, and in the model bacterium Deinococcus radiodurans, it is dispensable for radiation resistance. Many radiation-resistant organisms accumulate small-molecule Mn2+-antioxidant complexes well-known for their catalytic ability to scavenge O2•-, along with MnSOD, as exemplified by D. radiodurans. Here, we report experiments that relate the MnSOD and Mn-antioxidant content to aging and oxidative stress resistances and which indicate that C. elegans, like D. radiodurans, may rely on Mn-antioxidant complexes as the primary defense against reactive oxygen species (ROS). Wild-type and ΔMnSOD D. radiodurans and C. elegans were monitored for gamma radiation sensitivities over their life spans while gauging Mn2+-antioxidant content by electron paramagnetic resonance (EPR) spectroscopy, a powerful new approach to determining the in vivo Mn-antioxidant content of cells as they age. As with D. radiodurans, MnSOD is dispensable for radiation survivability in C. elegans, which hyperaccumulates Mn-antioxidants exceptionally protective of proteins. Unexpectedly, ΔMnSOD mutants of both the nematodes and bacteria exhibited increased gamma radiation survival compared to the wild-type. In contrast, the loss of MnSOD renders radiation-resistant bacteria sensitive to atmospheric oxygen during desiccation. Our results support the concept that the disparate responses to oxidative stress are explained by the accumulation of Mn-antioxidant complexes which protect, complement, and can even supplant MnSOD. IMPORTANCE The current theory of cellular defense against oxidative damage identifies antioxidant enzymes as primary defenders against ROS, with MnSOD being the preeminent superoxide (O2•-) scavenger. However, MnSOD is shown to be dispensable both for radiation resistance and longevity in model organisms, the bacterium Deinococcus radiodurans and the nematode Caenorhabditis elegans. Measured by electron paramagnetic resonance (EPR) spectroscopy, small-molecule Mn-antioxidant content was shown to decline in unison with age-related decreases in cell proliferation and radioresistance, which again are independent of MnSOD presence. Most notably, the Mn-antioxidant content of C. elegans drops precipitously in the last third of its life span, which links with reports that the steady-state level of oxidized proteins increases exponentially during the last third of the life span in animals. This leads us to propose that global responses to oxidative stress must be understood through an extended theory that includes small-molecule Mn-antioxidants as potent O2•--scavengers that complement, and can even supplant, MnSOD.

Chronic gamma radiation resistance in fungi correlates with resistance to chromium and elevated temperatures, but not with resistance to acute irradiation.

Exposure to chronic ionizing radiation (CIR) from nuclear power plant accidents, acts of terrorism, and space exploration poses serious threats to humans. Fungi are a group of highly radiation-resistant eukaryotes, and an understanding of fungal CIR resistance mechanisms holds the prospect of protecting humans. We compared the abilities of 95 wild-type yeast and dimorphic fungal isolates, representing diverse Ascomycota and Basidiomycota, to resist exposure to five environmentally-relevant stressors: CIR (long-duration growth under 36 Gy/h) and acute (10 kGy/h) ionizing radiation (IR), heavy metals (chromium, mercury), elevated temperature (up to 50 °C), and low pH (2.3). To quantify associations between resistances to CIR and these other stressors, we used correlation analysis, logistic regression with multi-model inference, and customized machine learning. The results suggest that resistance to acute IR in fungi is not strongly correlated with the ability of a given fungal isolate to grow under CIR. Instead, the strongest predictors of CIR resistance in fungi were resistance to chromium (III) and to elevated temperature. These results suggest fundamental differences between the mechanisms of resistance to chronic and acute radiation. Convergent evolution towards radioresistance among genetically distinct groups of organisms is considered here.

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.

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.

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.

Prospects for Fungal Bioremediation of Acidic Radioactive Waste Sites: Characterization and Genome Sequence of Rhodotorula taiwanensis MD1149.

Highly concentrated radionuclide waste produced during the Cold War era is stored at US Department of Energy (DOE) production sites. This radioactive waste was often highly acidic and mixed with heavy metals, and has been leaking into the environment since the 1950s. Because of the danger and expense of cleanup of such radioactive sites by physicochemical processes, in situ bioremediation methods are being developed for cleanup of contaminated ground and groundwater. To date, the most developed microbial treatment proposed for high-level radioactive sites employs the radiation-resistant bacterium Deinococcus radiodurans. However, the use of Deinococcus spp. and other bacteria is limited by their sensitivity to low pH. We report the characterization of 27 diverse environmental yeasts for their resistance to ionizing radiation (chronic and acute), heavy metals, pH minima, temperature maxima and optima, and their ability to form biofilms. Remarkably, many yeasts are extremely resistant to ionizing radiation and heavy metals. They also excrete carboxylic acids and are exceptionally tolerant to low pH. A special focus is placed on Rhodotorula taiwanensis MD1149, which was the most resistant to acid and gamma radiation. MD1149 is capable of growing under 66 Gy/h at pH 2.3 and in the presence of high concentrations of mercury and chromium compounds, and forming biofilms under high-level chronic radiation and low pH. We present the whole genome sequence and annotation of R. taiwanensis strain MD1149, with a comparison to other Rhodotorula species. This survey elevates yeasts to the frontier of biology's most radiation-resistant representatives, presenting a strong rationale for a role of fungi in bioremediation of acidic radioactive waste sites.