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.

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.

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.

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.

Complete Genome Sequence of the Cesium-Accumulating Bacterium Rhodococcus qingshengii CS98, Isolated from Soil in Japan.

Rhodococcus qingshengii CS98 is a bacterium isolated from soil in Japan that shows strong cesium-accumulating ability. Here, we report the complete genome sequence of R. qingshengii (6.7 Mb), which may provide useful genetic information supporting its bioremediation features.

DNA Damage and Survival Time Course of Deinococcal Cell Pellets During 3 Years of Exposure to Outer Space.

The hypothesis called "panspermia" proposes an interplanetary transfer of life. Experiments have exposed extremophilic organisms to outer space to test microbe survivability and the panspermia hypothesis. Microbes inside shielding material with sufficient thickness to protect them from UV-irradiation can survive in space. This process has been called "lithopanspermia," meaning rocky panspermia. We previously proposed sub-millimeter cell pellets (aggregates) could survive in the harsh space environment based on an on-ground laboratory experiment. To test our hypothesis, we placed dried cell pellets of the radioresistant bacteria Deinococcus spp. in aluminum plate wells in exposure panels attached to the outside of the International Space Station (ISS). We exposed microbial cell pellets with different thickness to space environments. The results indicated the importance of the aggregated form of cells for surviving in harsh space environment. We also analyzed the samples exposed to space from 1 to 3 years. The experimental design enabled us to get and extrapolate the survival time course to predict the survival time of Deinococcus radiodurans. Dried deinococcal cell pellets of 500 µm thickness were alive after 3 years of space exposure and repaired DNA damage at cultivation. Thus, cell pellets 1 mm in diameter have sufficient protection from UV and are estimated to endure the space environment for 2-8 years, extrapolating the survival curve and considering the illumination efficiency of the space experiment. Comparison of the survival of different DNA repair-deficient mutants suggested that cell aggregates exposed in space for 3 years suffered DNA damage, which is most efficiently repaired by the uvrA gene and uvdE gene products, which are responsible for nucleotide excision repair and UV-damage excision repair. Collectively, these results support the possibility of microbial cell aggregates (pellets) as an ark for interplanetary transfer of microbes within several years.

Draft Genome Sequence of Calcium-Dependent Novosphingobium sp. Strain TCA1, Isolated from a Hot Spring Containing a High Concentration of Calcium Ions.

Calcium-dependent Novosphingobium sp. strain TCA1 was newly isolated from a water sample from a hot spring containing a high concentration of calcium ions. Here, we report the draft genome sequence of this bacterium, which may be the basis for research on calcium ion homeostasis.

Complete Genome Sequence of a Radioresistant Bacterial Strain, Deinococcus grandis ATCC 43672.

Deinococcus grandis is a radioresistant bacterial species isolated from freshwater fish. In this article, we report the complete genome sequence of D. grandis strain ATCC 43672. This sequence is useful for comparative genomics to understand the traits of Deinococcus species and can be used as a reference in experimental genetics.

Sensitivity of Deinococcus grandis rodZ deletion mutant to calcium ions results in enhanced spheroplast size.

RodZ is a cytoskeletal protein associated with bacterial cell shape. It is a transmembrane protein located on the plasma membrane, and it binds to another cytoskeletal protein MreB. Deinococcus grandis contains a rodZ homolog. Although D. grandis is rod-shaped, it becomes spherical in shape when the rodZ homolog is disrupted. The rodZ deletion mutant was treated with lysozyme to generate spheroplasts. The spheroplasts enlarged in medium containing calcium chloride and penicillin. The rodZ deletion mutant spheroplasts were more sensitive to calcium ions than wild type. Cell and cytoplasm sizes of enlarged spheroplasts of the rodZ deletion mutant tended to be larger than those of wild type. Thus, disruption of rodZ enhances plasma and outer membrane expansion in D. grandis spheroplasts.

Pol B, a Family B DNA Polymerase, in Thermococcus kodakarensis is Important for DNA Repair, but not DNA Replication.

Thermococcus kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species.

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.

Draft Genome Sequence of the Radioresistant Bacterium Deinococcus aerius TR0125, Isolated from the High Atmosphere above Japan.

Deinococcus aerius strain TR0125 is a bacterium isolated from the high atmosphere above Japan that shows strong resistance to desiccation, UV-C, and gamma radiation. Here, we report the draft genome sequence of D. aerius (4.5 Mb), which may provide useful genetic information supporting its biochemical features.

Evaluation of metabolic changes in oxalate-rich plant Rumex obtusifolius L. caused by ion beam irradiation.

Some Rumex species such as sorrel are edible as baby leaf salad greens. On the other hand, Rumex plants accumulate soluble oxalate, a toxic metabolite which causes serious diseases such as renal syndrome. We attempted to produce low-oxalate plants of R. obtusifolius, a perennial weed which has higher vitamin C and amino acid content and higher tolerance to stress than many other Rumex species. Ion beams are ionising radiation with high linear energy transfer that causes a wide spectrum of mutations. Thus, in the present study we evaluated the effects of ion beams on oxalate and other primary metabolites in leaves of R. obtusifolius using CE-MS. The results showed that oxalate content was increased by irradiation with carbon ion beams. Metabolome analysis revealed that ion beams affected carbon flow to the isocitrate pathway, which is involved in oxalate synthesis. These observations suggested that modulation of carbon flow to the isocitrate pathway is important to regulate oxalate levels in plants.

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.

Draft Genome Sequence of the Radioresistant Bacterium Deinococcus grandis, Isolated from Freshwater Fish in Japan.

Deinococcus grandis is a radioresistant bacterium isolated from freshwater fish in Japan. Here we reported the draft genome sequence of D. grandis (4.1 Mb), which will be useful for elucidating the common principles of radioresistance in Deinococcus species through the comparative analysis of genomic sequences.

PprM, a Cold Shock Domain-Containing Protein from Deinococcus radiodurans, Confers Oxidative Stress Tolerance to Escherichia coli.

Escherichia coli is a representative microorganism that is frequently used for industrial biotechnology; thus its cellular robustness should be enhanced for the widespread application of E. coli in biotechnology. Stress response genes from the extremely radioresistant bacterium Deinococcus radiodurans have been used to enhance the stress tolerance of E. coli. In the present study, we introduced the cold shock domain-containing protein PprM from D. radiodurans into E. coli and observed that the tolerance to hydrogen peroxide (H2O2) was significantly increased in recombinant strains (Ec-PprM). The overexpression of PprM in E. coli elevated the expression of some OxyR-dependent genes, which play important roles in oxidative stress tolerance. Particularly, mntH (manganese transporter) was activated by 9-fold in Ec-PprM, even in the absence of H2O2 stress, which induced a more than 2-fold increase in the Mn/Fe ratio compared with wild type. The reduced production of highly reactive hydroxyl radicals (·OH) and low protein carbonylation levels (a marker of oxidative damage) in Ec-PprM indicate that the increase in the Mn/Fe ratio contributes to the protection of cells from H2O2 stress. PprM also conferred H2O2 tolerance to E. coli in the absence of OxyR. We confirmed that the H2O2 tolerance of oxyR mutants reflected the activation of the ycgZ-ymgABC operon, whose expression is activated by H2O2 in an OxyR-independent manner. Thus, the results of the present study showed that PprM could be exploited to improve the robustness of E. coli.

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.

Draft Genome Sequence of Methylobacterium sp. ME121, Isolated from Soil as a Mixed Single Colony with Kaistia sp. 32K.

Methylobacterium sp. ME121 was isolated from soil as a mixed single colony with Kaistia sp. 32K, and its growth was enhanced by coculture. Here, we report the draft genome sequence of Methylobacterium sp. ME121, which may contribute to the study of the molecular mechanisms underlying this phenomenon.