Hirofumi Ohba

Deinococcus aerius sp. nov., isolated from the high atmosphere

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.

Deinococcus aetherius sp. nov., isolated from the stratosphere.

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)).

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination.

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.

Identification of PprM: a modulator of the PprI-dependent DNA damage response in Deinococcus radiodurans

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 γ-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 pprApprM 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.

Problems with the current deinococcal hypothesis: an alternative theory.

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”.

Development of versatile shuttle vectors for Deinococcus grandis

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.