Hirofumi Ohba

Japan Atomic Energy Agency, Muramatsu, Niigata, Japan

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Publications (13)21.5 Total impact

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    ABSTRACT: Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed.
    DNA repair 02/2012; 11(4):410-8. · 4.20 Impact Factor
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    ABSTRACT: The moderately thermophilic bacterium Deinococcus geothermalis exhibits extraordinary resistance to ionizing radiation. RecA protein is considered to be one of the most important participants in radioresistance. To assess the role of the RecA protein in D. geothermalis, the recA gene was isolated from D. geothermalis and over expressed in Escherichia coli. After the D. geothermalis RecA protein (GeoRecA) was purified, the recombination activity was investigated in vitro. GeoRecA efficiently promoted the strand exchange reaction between homologous linear double-stranded DNA and circular single-stranded DNA substrates at 50°C. Like Deinococcus radiodurans RecA protein (DraRecA), GeoRecA could promote DNA strand exchange reaction through normal and inverse pathways. Furthermore, GeoRecA complemented the RecA deficiency of D. radiodurans. These results indicate that GeoRecA is a functional homologue of DraRecA and plays an important role in radioresistance. However, unlike DraRecA, GeoRecA could not complement the RecA deficiency of E. coli, suggesting that GeoRecA require more strict intracellular conditions than DraRecA does to fulfill its function. This study provides new insights into the role of deinococcal RecA protein in radioresistance.
    African Journal of Biochemistry Research. 05/2010; 4:111-118.
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    ABSTRACT: The moderately thermophilic bacterium Deinococcus geothermalis exhibits extraordinary resistance to ionizing radiation. RecA protein is considered to be one of the most important participants in radioresistance. To assess the role of the RecA protein in D. geothermalis, the recA gene was isolated from D. geothermalis and over expressed in Escherichia coli. After the D. geothermalis RecA protein (GeoRecA) was purified, the recombination activity was investigated in vitro. GeoRecA efficiently promoted the strand exchange reaction between homologous linear double-stranded DNA and circular single-stranded DNA substrates at 50°C. Like Deinococcus radiodurans RecA protein (DraRecA), GeoRecA could promote DNA strand exchange reaction through normal and inverse pathways. Furthermore, GeoRecA complemented the RecA deficiency of D. radiodurans. These results indicate that GeoRecA is a functional homologue of DraRecA and plays an important role in radioresistance. However, unlike DraRecA, GeoRecA could not complement the RecA deficiency of E. coli, suggesting that GeoRecA require more strict intracellular conditions than DraRecA does to fulfill its function. This study provides new insights into the role of deinococcal RecA protein in radioresistance.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 04/2010; 4:111-8. · 0.57 Impact Factor
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    ABSTRACT: A pink-red pigmented, non-motile, coccoid bacterial strain, ST0316(T), was isolated from dust samples collected from the stratosphere in Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that it belonged to the genus Deinococcus. DNA G+C content (69.8 mol%), desiccation tolerance, and resistance to gamma-rays [D(10) (dose required to reduce the bacterial population by 10-fold) >8 kGy] and UV radiation (D(10) 1000 J m(-2)) supported the affiliation of strain ST0316(T) to the genus Deinococcus. The major peptidoglycan amino acids were d-glutamic acid, glycine, d-alanine, l-alanine and ornithine. Predominant fatty acids were C(16 : 1)omega7c, C(16 : 0), C(17 : 0) and iso-C(17 : 0). Strain ST0316(T) diverged from recognized species of the genus Deinococcus, showing less than 93.0 % similarity values to its closest relatives Deinococcus apachensis, D. aerius, D. geothermalis and D. murrayi. Strain ST0316(T) also differed from the type strains of closely related species in its polar lipid profile, nitrate reduction and carbon-source assimilation tests. Therefore, we propose a new species of the genus Deinococcus, Deinococcus aetherius sp. nov. (type strain, ST0316(T) =JCM 11751(T) =DSM 21230(T)).
    International journal of systematic and evolutionary microbiology 08/2009; 60(Pt 4):776-9. · 2.11 Impact Factor
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    ABSTRACT: An orange-pigmented, non-motile, coccoid bacterial strain, designated TR0125T, was isolated from dust samples collected in the high atmosphere above Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was within the radiation of Deinococcus species. The major peptidoglycan amino acids were D-glutamic acid, glycine, D-alanine, L-alanine and ornithine. The predominant fatty acids were iso-C17:0, iso-C17:1omega9c and iso-C15:0. Strong resistance to desiccation, UV-C and gamma radiation and high DNA G+C content also supported the affiliation of strain TR0125T to the genus Deinococcus. Strain TR0125T showed the highest 16S rRNA gene sequence similarity value (95.7%) to the type strain of Deinococcus apachensis, and phylogenetic analysis showed that it was further separated from D. apachensis than from Deinococcus geothermalis, indicating that strain TR0125T was not a member of these two Deinococcus species. In addition, phenotypic differences were found between strain TR0125T and the type strains of these two Deinococcus species. Therefore, a novel species of the genus Deinococcus, Deinococcus aerius sp. nov. (type strain, TR0125T=JCM 11750T=DSM 21212T), is proposed to accommodate this isolate.
    International journal of systematic and evolutionary microbiology 07/2009; 59(Pt 8):1862-6. · 2.11 Impact Factor
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    ABSTRACT: To develop new shuttle vectors for Deinococcus species, the nucleotide sequence of the small cryptic plasmid pUE30 from Deinococcus radiopugnans ATCC19172 was determined. The 2467-bp plasmid possesses two open reading frames, one encoding 88 amino acid residues (Orf1) and the other encoding 501 amino acid residues (Orf2). The predicted amino acid sequence encoded by Orf1 exhibits similarity to the N-terminal regions of replication proteins encoded by repABC-type plasmids of a-proteobacteria. On the other hand, the predicted amino acid sequence encoded by Orf2 exhibits similarity to replication proteins encoded by plasmids of D. radiodurans SARK and Thermus species. Hybrid plasmids consisting of pUE30 and pKatCAT5, which replicates in E. coli with a chloramphenicol resistance determinant, were shown to autonomously replicate in D. grandis ATCC43672. Deletion analysis revealed that Orf2 was necessary for replication of the plasmids in D. grandis. On the other hand, a DNA fragment encompassing the Orf1-coding region was involved in the instability of the plasmid in D. grandis. An expression plasmid that possesses the D. radiodurans minimal groE promoter was constructed, and a firefly luciferase gene was successfully expressed in D. grandis. The D. grandis host-vector system developed in this study should prove useful in the bioremediation of radioactive waste and for the investigation of DNA repair mechanisms.
    Plasmid 07/2009; 62(1):1-9. · 1.76 Impact Factor
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    ABSTRACT: Locomotory behavior (motility) and mechanosensation are of vital importance in animals. We examined the effects of ionizing radiation (IR) on locomotory behavior and mechanosensation using a model organism, the nematode Caenorhabditis elegans. Bacterial mechanosensation in C. elegans induces the dopamine-mediated slowing of locomotion in the presence of bacteria (food), known as the basal slowing response. We previously reported an IR-induced reduction of locomotory rate in the absence of food. In the present study, we observed a similar IR-induced reduction of locomotory rate in the cat-2 mutant, which is defective in bacterial mechanosensation. The dose response pattern of the locomotory rate in the presence of food was relatively flat in wild-type animals, but not in cat-2 mutants. This suggests that the dopamine system, which is related to bacterial mechanosensation in C. elegans, might have a dominant effect on locomotory rate in the presence of food, which masks the effects of other stimuli. Moreover, we found that the behavioral responses of hydrogen peroxide-exposed wild-type animals are similar to those of IR-exposed animals. Our findings suggest that the IR-induced reduction of locomotory rate in the absence of food is mediated by a different pathway from that for bacterial mechanosensation, at least partially through IR-produced hydrogen peroxide.
    Journal of Radiation Research 03/2009; 50(2):119-25. · 1.45 Impact Factor
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    ABSTRACT: Deinococcus radiodurans possesses a DNA damage response mechanism that acts via the PprI protein to induce RecA and PprA proteins, both of which are necessary in conferring extreme radioresistance. In an effort to further delineate the nature of the DNA damage response mechanism in D. radiodurans, we set out to identify novel components of the PprI-dependent signal transduction pathway in response to radiation stress. Here we demonstrate the discovery of a novel regulatory protein, PprM (a modulator of the PprI-dependent DNA damage response), which is a homolog of cold shock protein (Csp). Disruption of the pprM gene rendered D. radiodurans significantly sensitive to gamma-rays. PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from D. geothermalis and Thermus thermophilus. Purified PprM is present as a homodimer under physiological conditions, as the case with Escherichia coli CspD. The pprA pprM double-disruptant strain exhibited higher sensitivity than the pprA or pprM single disruptant strains, suggesting that PprM regulates other hitherto unknown protein(s) important for radioresistance besides PprA. This study strongly suggests that PprM is involved in the radiation response mediated by PprI in D. radiodurans.
    Extremophiles 03/2009; 13(3):471-9. · 2.20 Impact Factor
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    ABSTRACT: All theories related to the evolution of Deinococcus radiodurans have a common denominator: the strong positive correlation between ionizing-radiation resistance and desiccation tolerance. Currently, the widespread hypothesis is that D. radiodurans' ionizing-radiation resistance is a consequence of this organism's adaptation to desiccation (desiccation adaptation hypothesis). Here, we draw attention to major discrepancy that has emerged between the "desiccation adaptation hypothesis" and recent findings in computational biology, experimental research, and terrestrial subsurface surveys. We explain why the alternative hypothesis, suggesting that D. radiodurans' desiccation tolerance could be a consequence of this organism's adaptation to ionizing radiation (radiation adaptation hypothesis), should be considered on equal basis with the "desiccation adaptation hypothesis".
    Theory in Biosciences 09/2007; 126(1):43-5. · 0.93 Impact Factor
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    ABSTRACT: The extremely radioresistant bacterium Deinococcus radiodurans contains two LexA homologues (LexA1 and LexA2) that are possible transcriptional regulators associated with the DNA damage response. In this study, resequencing revealed that there was an additional cytosine nucleotide (nucleotide position 612) in the D. radiodurans lexA2 gene. Purified LexA2 possessed proteolytic activity that could be stimulated by RecA. In an effort to gain an insight into the role of LexA2 in the radiation response mechanism, recA, lexA1 and lexA2 disruptant strains were generated and investigated. The intracellular level of RecA increased in lexA1 and lexA2 disruptant strains following gamma-irradiation as in the wild-type strain. These results indicated that the two LexA homologues did not possess functional overlap regarding the induction of RecA. The lexA2 disruptant strains exhibited a much higher resistance to gamma-rays than the wild-type strain. Furthermore, a luciferase assay showed that pprA promoter activation was enhanced in the lexA2 disruptant strain following gamma-irradiation. The pprA gene encoding the novel radiation-inducible protein PprA plays a critical role in the radioresistance of D. radiodurans. The increase in radioresistance of the lexA2 disruptant strain is explained in part by the enhancement of pprA promoter activation.
    Microbiology 12/2006; 152(Pt 11):3217-26. · 2.85 Impact Factor
  • Katsuya SATOH, Hirofumi OHBA, Issay NARUMI
    Seibutsu Butsuri. 01/2006; 46(5):270-274.
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    ABSTRACT: In a previous study, we identified a novel radiation-inducible protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans [Narumi, I., Satoh, K., Cui, S., Funayama, T., Kitayama, S., Watanabe, H., 2004. PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol. Microbiol. 54, 278-285.]. Despite the interest in mechanisms underlying radiation responses in D. radiodurans, little is known about the radiation responsive promoter for radiation-inducible proteins. In this study, three transcriptional start points for pprA mRNA were identified by primer extension analysis, located at positions -156, -154 and -22 upstream from the pprA translation initiation site. The amount of the three extended products increased in cells exposed to 2 kGy followed by a 0.5-h post-incubation. This suggested the existence of at least two radiation responsive promoters for pprA expression. Functional characterization of the upstream region of the pprA gene using a luciferase reporter assay revealed that the distal promoter is located between positions -208 and -156 from the translation initiation site, while the proximal promoter is located between positions -57 and -22. The region located between positions -57 and -38 was indispensable for proximal promoter activity. Site-directed mutagenesis of a thymine positioned at -33 resulted in severe impairment of promoter activity, and suggested that the thymine functions as a master base for the proximal radiation responsive promoter. The product of the D. radiodurans pprI gene is thought to be a general switch in the radiation response [Hua, Y., Narumi, I., Gao, G., Tian, B., Satoh, K., Kitayama, S., Shen, B., 2003. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem. Biophys. Res. Commun. 306, 354-360.]. We examined the effect of pprI disruption on pprA promoter activity. The results suggested that up-regulation of pprA expression by the pprI gene product is triggered at the promoter level.
    Gene 01/2006; 363:133-41. · 2.20 Impact Factor
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    ABSTRACT: The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.
    Genes & Genetic Systems 11/2003; 78(5):319-27. · 1.13 Impact Factor