Microbiome analysis of the restricted bacteria in radioactive element-containing water at the Fukushima Daiichi Nuclear Power Station.

A major incident occurred at the Fukushima Daiichi Nuclear Power Station following the tsunami triggered by the Tohoku-Pacific Ocean Earthquake in March 2011, whereby seawater entered the torus room in the basement of the reactor building. Here, we identify and analyze the bacterial communities in the torus room water and several environmental samples. Samples of the torus room water (1 × 109 Bq137Cs/L) were collected by the Tokyo Electric Power Company Holdings from two sampling points between 30 cm and 1 m from the bottom of the room (TW1) and the bottom layer (TW2). A structural analysis of the bacterial communities based on 16S rRNA amplicon sequencing revealed that the predominant bacterial genera in TW1 and TW2 were similar. TW1 primarily contained the genus Limnobacter, a thiosulfate-oxidizing bacterium. γ-Irradiation tests on Limnobacter thiooxidans, the most closely related phylogenetically found in TW1, indicated that its radiation resistance was similar to ordinary bacteria. TW2 predominantly contained the genus Brevirhabdus, a manganese-oxidizing bacterium. Although bacterial diversity in the torus room water was lower than seawater near Fukushima, ~70% of identified genera were associated with metal corrosion. Latent environment allocation-an analytical technique that estimates habitat distributions and co-detection analyses-revealed that the microbial communities in the torus room water originated from a distinct blend of natural marine microbial and artificial bacterial communities typical of biofilms, sludge, and wastewater. Understanding the specific bacteria linked to metal corrosion in damaged plants is important for advancing decommissioning efforts. IMPORTANCE: In the context of nuclear power station decommissioning, the proliferation of microorganisms within the reactor and piping systems constitutes a formidable challenge. Therefore, the identification of microbial communities in such environments is of paramount importance. In the aftermath of the Fukushima Daiichi Nuclear Power Station accident, microbial community analysis was conducted on environmental samples collected mainly outside the site. However, analyses using samples from on-site areas, including adjacent soil and seawater, were not performed. This study represents the first comprehensive analysis of microbial communities, utilizing meta 16S amplicon sequencing, with a focus on environmental samples collected from the radioactive element-containing water in the torus room, including the surrounding environments. Some of the identified microbial genera are shared with those previously identified in spent nuclear fuel pools in countries such as France and Brazil. Moreover, our discussion in this paper elucidates the correlation of many of these bacteria with metal corrosion.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Enlargement of Deinococcus grandis spheroplasts requires Mg2+ or Ca2.

While the cell wall strictly controls cell size and morphology in bacteria, spheroplasts lack cell walls and can become enlarged in growth medium under optimal conditions. Optimal conditions depend on the bacterial species. We frequently observed extreme enlargement of spheroplasts of the radiation-resistant bacterium Deinococcus grandis in Difco Marine Broth 2216, but not in TGY broth (a commonly used growth medium for Deinococcus). Thorough investigation of media components showed that the presence of Mg2+ or Ca2+ promoted extreme spheroplast enlargement, synthesizing the outer membrane. Our findings strongly suggest that Mg2+ or Ca2+ enlarges spheroplasts, which could change the lipid composition of the spheroplast membrane.

Arabidopsis pab1, a mutant with reduced anthocyanins in immature seeds from banyuls, harbors a mutation in the MATE transporter FFT.

Forward genetics approaches have helped elucidate the anthocyanin biosynthetic pathway in plants. Here, we used the Arabidopsis banyuls (ban) mutant, which accumulates anthocyanins, instead of colorless proanthocyanidin precursors, in immature seeds. In contrast to standard screens for mutants lacking anthocyanins in leaves/stems, we mutagenized ban plants and screened for mutants showing differences in pigmentation of immature seeds. The pale banyuls1 (pab1) mutation caused reduced anthocyanin pigmentation in immature seeds compared with ban. Immature pab1 ban seeds contained less anthocyanins and flavonols than ban, but showed normal expression of anthocyanin biosynthetic genes. In contrast to pab1, introduction of a flavonol-less mutation into ban did not produce paler immature seeds. Map-based cloning showed that two independent pab1 alleles disrupted the MATE-type transporter gene FFT/DTX35. Complementation of pab1 with FFT confirmed that mutation in FFT causes the pab1 phenotype. During development, FFT promoter activity was detected in the seed-coat layers that accumulate flavonoids. Anthocyanins accumulate in the vacuole and FFT fused to GFP mainly localized in the vacuolar membrane. Heterologous expression of grapevine MATE-type anthocyanin transporter gene partially complemented the pab1 phenotype. These results suggest that FFT acts at the vacuolar membrane in anthocyanin accumulation in the Arabidopsis seed coat, and that our screening strategy can reveal anthocyanin-related genes that have not been found by standard screening.

DNA Gyrase of Deinococcus radiodurans is characterized as Type II bacterial topoisomerase and its activity is differentially regulated by PprA in vitro.

The multipartite genome of Deinococcus radiodurans forms toroidal structure. It encodes topoisomerase IB and both the subunits of DNA gyrase (DrGyr) while lacks other bacterial topoisomerases. Recently, PprA a pleiotropic protein involved in radiation resistance in D. radiodurans has been suggested for having roles in cell division and genome maintenance. In vivo interaction of PprA with topoisomerases has also been shown. DrGyr constituted from recombinant gyrase A and gyrase B subunits showed decatenation, relaxation and supercoiling activities. Wild type PprA stimulated DNA relaxation activity while inhibited supercoiling activity of DrGyr. Lysine133 to glutamic acid (K133E) and tryptophane183 to arginine (W183R) replacements resulted loss of DNA binding activity in PprA and that showed very little effect on DrGyr activities in vitro. Interestingly, wild type PprA and its K133E derivative continued interacting with GyrA in vivo while W183R, which formed relatively short oligomers did not interact with GyrA. The size of nucleoid in PprA mutant (1.9564 ± 0.324 µm) was significantly bigger than the wild type (1.6437 ± 0.345 µm). Thus, we showed that DrGyr confers all three activities of bacterial type IIA family DNA topoisomerases, which are differentially regulated by PprA, highlighting the significant role of PprA in DrGyr activity regulation and genome maintenance in D. radiodurans.

Developing a new host-vector system for Deinococcus grandis.

Deinococcus spp. are known for their radiation resistance, toxic compound removal, and production of valuable substances. Therefore, developing gene expression systems for Deinococcus spp. is crucial in advancing genetic engineering applications. To date, plasmid vectors that express foreign genes in D. radiodurans and D. geothermalis have been limited to plasmid pI3 and its derivatives. In contrast, plasmid vectors that express foreign genes in D. grandis include plasmid pZT23 and its derivatives. In this study, we developed a new system for the stable introduction and retention of expression plasmids for D. grandis. Two cryptic plasmids were removed from the wild-type strain to generate the TY3 strain. We then constructed a shuttle vector plasmid, pGRC5, containing the replication initiation region of the smallest cryptic plasmid, pDEGR-3, replication initiation region of the E. coli vector, pACYC184, and an antibiotic resistance gene. We introduced pGRC5, pZT23-derived plasmid pZT29H, and pI3-derived plasmid pRADN8 into strain TY3, and found their coexistence in D. grandis cells. The quantitative PCR assay results found that pGRC5, pZT29H, and pRADN8 had relative copy numbers of 11, 26, and 5 per genome, respectively. Furthermore, we developed a new plasmid in which the luciferase gene was controlled by the promoter region, which contained radiation-desiccation response operator sequences for D. grandis DdrO, a stress response regulon repressor in D. grandis, hence inducing gene expression via ultraviolet-C light irradiation. These plasmids are expected to facilitate the removal and production of toxic and valuable substances, in D. grandis, respectively, particularly of those involving multiple genes.

Effect of gamma irradiation on the proteogenome of cold-acclimated Kocuria rhizophila PT10.

The effects of ionizing radiation (IR) on the protein dynamics of cold-stressed cells of a radioresistant actinobacterium, Kocuria rhizophila PT10, isolated from the rhizosphere of the desert plant Panicum turgidum were investigated using a shotgun methodology based on nanoflow liquid chromatography coupled to tandem mass spectrometry. Overall, 1487 proteins were certified, and their abundances were compared between the irradiated condition and control. IR of cold-acclimated PT10 triggered the over-abundance of proteins involved in (1) a strong transcriptional regulation, (2) amidation of peptidoglycan and preservation of cell envelope integrity, (3) detoxification of reactive electrophiles and regulation of the redox status of proteins, (4) base excision repair and prevention of mutagenesis and (5) the tricarboxylic acid (TCA) cycle and production of fatty acids. Also, one of the more significant findings to emerge from this study is the SOS response of stressed PT10. Moreover, a comparison of top hits radio-modulated proteins of cold-acclimated PT10 with proteomics data from gamma-irradiated Deinococcus deserti showed that stressed PT10 has a specific response characterised by a high over-abundance of NemA, GatD, and UdgB.

SMALL ACIDIC PROTEIN1 acts with RUB modification components, the COP9 signalosome, and AXR1 to regulate growth and development of Arabidopsis.

Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.

Deinococcus radiodurans YgjD and YeaZ are involved in the repair of DNA cross-links.

Orthologs of Escherichia coli ygjD and yeaZ genes are highly conserved in various organisms. The genome of the radioresistant bacterium Deinococcus radiodurans possesses single orthologs of ygjD (DR_0382) and yeaZ (DR_0756). Complete loss of either one or both genes did not result in any significant changes in cell growth efficiency, indicating that both genes are not essential for cell viability in D. radiodurans, unlike the case with other species such as E. coli, Bacillus subtilis and Saccharomyces cerevisiae. Survival rates following DNA damage induced by hydrogen peroxide (H(2)O(2)), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ultra violet (UV) radiation, γ-rays, cisplatin and mitomycin C (MMC) were compared among the wild-type strain and D. radiodurans ygjD/yeaZ null mutants. Cell viability of the null mutants did not decrease following exposure to H(2)O(2) or MNNG. In addition, the reduction in cell viability following exposure to γ-rays, UV radiation or cisplatin was marginal in the null mutants compared to the wild-type strain. Interestingly, the null mutants exhibited high sensitivity to MMC, which mainly causes interstrand DNA cross-links. The sensitivity of the null mutants to MMC was restored to that of the wild type by transformation with plasmids expressing these genes. These results suggest that D. radiodurans ygjD and yeaZ genes are involved in DNA repair and play a role in the repair of DNA cross-links.

The possible interplanetary transfer of microbes: assessing the viability of Deinococcus spp. under the ISS Environmental conditions for performing exposure experiments of microbes in the Tanpopo mission.

To investigate the possible interplanetary transfer of life, numerous exposure experiments have been carried out on various microbes in space since the 1960s. In the Tanpopo mission, we have proposed to carry out experiments on capture and space exposure of microbes at the Exposure Facility of the Japanese Experimental Module of the International Space Station (ISS). Microbial candidates for the exposure experiments in space include Deinococcus spp.: Deinococcus radiodurans, D. aerius and D. aetherius. In this paper, we have examined the survivability of Deinococcus spp. under the environmental conditions in ISS in orbit (i.e., long exposure to heavy-ion beams, temperature cycles, vacuum and UV irradiation). A One-year dose of heavy-ion beam irradiation did not affect the viability of Deinococcus spp. within the detection limit. Vacuum (10(-1) Pa) also had little effect on the cell viability. Experiments to test the effects of changes in temperature from 80 °C to -80 °C in 90 min (± 80 °C/90 min cycle) or from 60 °C to -60 °C in 90 min (± 60 °C/90 min cycle) on cell viability revealed that the survival rate decreased severely by the ± 80 °C/90 min temperature cycle. Exposure of various thicknesses of deinococcal cell aggregates to UV radiation (172 nm and 254 nm, respectively) revealed that a few hundred micrometer thick aggregate of deinococcal cells would be able to withstand the solar UV radiation on ISS for 1 year. We concluded that aggregated deinococcal cells will survive the yearlong exposure experiments. We propose that microbial cells can aggregate as an ark for the interplanetary transfer of microbes, and we named it 'massapanspermia'.

Role of AtPolζ, AtRev1, and AtPolη in UV light-induced mutagenesis in Arabidopsis.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism in which DNA lesions are bypassed by specific polymerases. To investigate the role of TLS activities in ultraviolet light-induced somatic mutations, we analyzed Arabidopsis (Arabidopsis thaliana) disruptants of AtREV3, AtREV1, and/or AtPOLH genes that encode TLS-type polymerases. The mutation frequency in rev3-1 or rev1-1 mutants decreased compared with that in the wild type, suggesting that AtPolζ and AtRev1 perform mutagenic bypass events, whereas the mutation frequency in the polh-1 mutant increased, suggesting that AtPolη performs nonmutagenic bypass events with respect to ultraviolet light-induced lesions. The rev3-1 rev1-1 double mutant showed almost the same mutation frequency as the rev1-1 single mutant. The increased mutation frequency found in polh-1 was completely suppressed in the rev3-1 polh-1 double mutant, indicating that AtPolζ is responsible for the increased mutations found in polh-1. In summary, these results suggest that AtPolζ and AtRev1 are involved in the same (error-prone) TLS pathway that is independent from the other (error-free) TLS pathway mediated by AtPolη.

Mutagenic effects of carbon ions near the range end in plants.

To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/µm) were compared with the effects of carbon ions penetrating the seeds (mean LET: 113keV/µm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/µm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>∼30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/µm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/µm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.

Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae.

Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was (12)C(5+) ion beams with an LET of 121keV/µm. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/µm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/µm were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67×10(-3)) at 400Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between (12)C(5+) ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation.

Development of a simple multiple mutation detection system using seed-coat flavonoid pigments in irradiated Arabidopsis M1 plants.

Ionizing radiation induces genetic variations in plants, which makes it useful for plant breeding. A theory that the induced mutations occur randomly in the genome has long been accepted, but is now controversial. Nevertheless, a comparative analysis of the mutations at multiple loci has not been conducted using irradiated M1 genomes that contain all types of mutations. In this study, we identified Arabidopsis mutants (pab2 and pab3) in a mutagenized population of an anthocyanin-positive seed mutant (ban). Both pab2 and pab3 were revealed to be double mutants (tt4 ban and tt8 ban, respectively) that produced similar anthocyanin-less immature seeds, but differentially colored mature seeds. These features enabled the seed color-based detection of de novo M1 mutations in TT4 or TT8 following the irradiation of double heterozygous plants (TT4/tt4 TT8/tt8 ban/ban). Most of the irradiated double heterozygous plants produced anthocyanin-positive immature seeds, but 19 plants produced anthocyanin-less immature seeds. Of these 19 mutants, 2 and 17 exhibited tt4- and tt8-type mature seed coloration, respectively. The molecular analysis of the seed coat DNA from randomly selected anthocyanin-less seeds detected mutations at the locus predicted on the basis of the phenotype. Thus, the simple system developed in this study can reliably detect radiation-induced mutations at multiple loci in irradiated Arabidopsis M1 plants.

Complete Genome Sequence of the Radioresistant Bacterium Deinococcus aetherius ST0316, Isolated from Air Dust Collected in the Lower Stratosphere above Japan.

Deinococcus aetherius ST0316 is a radioresistant bacterium that possess proficient DNA repair capacity. Here, we report the complete genome sequence of D. aetherius, which was obtained by hybrid assembly using short- and long-read sequencing. This sequence will be important information for elucidating the unique DNA repair mechanism of Deinococcus bacteria.

Metabolic profiling and cytological analysis of proanthocyanidins in immature seeds of Arabidopsis thaliana flavonoid accumulation mutants.

Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione-S-transferase (GST)-like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar-like structures, whereas those in tt12, a mutant of the tonoplast-bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP-TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent-insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent-soluble fraction by LC-MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP-TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.

Isolation and characterization of the fragrant cyclamen O-methyltransferase involved in flower coloration.

Anthocyanin O-methyltransferase (OMT) is one of the key enzymes for anthocyanin modification and flower pigmentation. We previously bred a novel red-purple-flowered fragrant cyclamen (KMrp) from the purple-flowered fragrant cyclamen 'Kaori-no-mai' (KM) by ion-beam irradiation. Since the major anthocyanins in KMrp and KM petals were delphinidin 3,5-diglucoside and malvidin 3,5-diglucoside, respectively, inactivation of a methylation step in the anthocyanin biosynthetic pathway was indicated in KMrp. We isolated and compared OMT genes expressed in KM and KMrp petals. RT-PCR analysis revealed that CkmOMT2 was expressed in the petals of KM but not in KMrp. Three additional CkmOMTs with identical sequences were expressed in petals of both KM and KMrp. Genomic PCR analysis revealed that CkmOMT2 was not amplified from the KMrp genome, indicating that ion-beam irradiation caused a loss of the entire CkmOMT2 region in KMrp. In vitro enzyme assay demonstrated that CkmOMT2 catalyzes the 3' or 3',5' O-methylation of the B-ring of anthocyanin substrates. These results suggest that CkmOMT2 is functional for anthocyanin methylation, and defective expression of CkmOMT2 is responsible for changes in anthocyanin composition and flower coloration in KMrp.

Mutation Analysis of the rpoB Gene in the Radiation-Resistant Bacterium Deinococcus radiodurans R1 Exposed to Space during the Tanpopo Experiment at the International Space Station.

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium Deinococcus radiodurans were exposed to various space environmental conditions at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission. Mutation analysis was done by sequencing the rpoB gene encoding RNA polymerase ß-subunit of the rifampicin-resistant mutants. Samples included bacteria exposed to the space environment with and without exposure to UV radiation as well as control samples held in the ISS cabin and at ground. The mutation sites of the rpoB gene obtained from the space-exposed and ISS/ground control samples were similar to the rpoB mutation sites previously reported in D. radiodurans. Most mutations were found at or near the rifampicin binding site in the RNA polymerase ß-subunit. Mutation sites found in UV-exposed samples were mostly shared with non-exposed and ISS/ground control samples. These results suggest that most mutations found in our experiments were induced during procedures that were applied across all treatments: preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. Some mutations may be enhanced by specific factors in the space experiments, but the mutations were also found in the spontaneous and control samples. Our experiment suggests that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. However, upon arrival at a recipient location, they must still be able to survive and repair the general damage induced during travel.

A UVB-hypersensitive mutant in Arabidopsis thaliana is defective in the DNA damage response.

To investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to gamma-radiation and hydroxyurea. This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr. The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1. RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons. The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region. Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast two-hybrid system, suggesting that the proteins form a homodimer. Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after gamma-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage. Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP.

Purification, crystallization and preliminary X-ray diffraction analysis of DNA damage response A protein from Deinococcus radiodurans.

DNA damage response A protein (DdrA) from Deinococcus radiodurans has been suggested to be involved in DNA-repair processes through binding to 3'-ends of single-stranded DNA, thereby protecting the ends from nuclease digestion. In this study, a recombinant C-terminally truncated form of D. radiodurans DdrA (DdrA157) comprising the first 157 residues of DdrA was expressed in Escherichia coli, purified and crystallized. Single crystals of DdrA157 were obtained by the hanging-drop method at 293 K. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a=46.31, b=180.26, c=114.17 Å, ß=90.02°. The crystal was expected to contain 14 molecules in the asymmetric unit. Diffraction data were collected to 2.35 Šresolution on beamline BL-5 at Photon Factory and initial phase determinations were attempted by the molecular-replacement method using the human Rad52 structure.