The method of electrophoretic mobility shift assay under high-pressure conditions was improved using a high-pressure electrophoresis apparatus with capillary narrow-tube gel. It was found that the protein-DNA complex in the gel was stained as a high-resolution spot with ethidium bromide. Using this method, it was found that the behavior under high-pressure conditions of the protein-DNA complex composed of NtrC protein and its target promoter DNA is important for the pressure-regulated transcription process, and it was confirmed that the complex was dissociated above a pressure of 70 MPa.

The rpoE gene encoding an RNA polymerase sigmaE subunit was isolated from a gamma-phage library of the deep-sea piezophilic and psychrophilic bacterium Shewanella violacea strain DSS12. Structual analysis showed that the gene organization of the fragment containing S. violacearpoE was the l-aspartate oxidase-coding gene, rpoE, rseA, rseB and rseC in that order, the same as in the case of Photobacterium profundum SS9 and Escherichia coli K-12. The cloned gene, 576 bp in length, was found to encode a protein consisting of 192 amino acid residues with a molecular mass of 21,806 Da. Amino acid alignment of the RpoE protein showed that the functional domains responsible for DNA recognition, DNA melting, core binding, and RseA interaction were highly conserved. We purified hexahistidine-fused RpoE protein by constructing an overexpression plasmid. Core-binding analysis revealed that the cloned RpoE protein has the ability to bind with core RNA polymerase as a sigma factor.

RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was sigma(70) containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the beta and beta' subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni(2+) affinity column. In vitro reconstitution using all proteins for the holoenzyme (alpha, beta, beta', sigma(70)) was carried out and the activity of the recombinant RNA polymerase was detected.

RNA polymerase was purified from the piezophile Shewanella violacea DSS12, and the transcriptional activity after pressure treatment was compared with that of the mesophile Escherichia coli. Application of pressure at 100 MPa for 30 min reduced the E. coli RNA polymerase activity to 60% of the activity at atmospheric pressure, whereas the S. violacea RNA polymerase maintained full activity, indicating that the S. violacea RNA polymerase is more stable than its E. coli counterpart. This result was supported by the analysis of the strength of subunit interactions of the enzyme from both species, using a high-pressure electrophoresis apparatus, which showed that a pressure of 140 MPa caused dissociation of E. coli RNA polymerase but not that of S. violacea RNA polymerase. On the other hand, the core enzyme of S. violacea RNA polymerase, which lacked the sigma70 factor, was dissociated at 140 MPa. These results suggest that the sigma70 factor is required for stabilization of S. violacea RNA polymerase under high-pressure conditions. In this paper, we provide in vitro evidence for piezoadaptation at the transcriptional level, using purified RNA polymerase from cells of S. violacea and E. coli.

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.

Deep-sea bacteria have unique systems for gene and protein expression controlled by hydrostatic pressure. One of the sigma factors, sigma54, was found to play an important role in pressure-regulated transcription in a deep-sea piezophilic bacterium, Shewanella violacea. A glutamine synthetase gene (glnA) has been targeted as a model for the pressure-regulated promoter to investigate transcriptional regulation by the sigma54 factor. Recognition sites for sigma54 and sigma70 factors were observed at an upstream region of the glnA, and NtrC-binding sites were also identified at the same region. Primer extension analyses revealed that the transcription initiation sites of both promoters were determined and that transcription from the sigma54 site was regulated by elevated pressure. The sigma54 promoter is known to be activated by a two-component signal transduction system, the NtrB-NtrC phosphorylation relay. Our results suggested that this system might be regulated by deep-sea conditions and that the gene expression controlled by the sigma54 promoter was actually regulated by pressure. We propose a possible model of the molecular mechanisms for pressure-regulated transcription.

Two genes for alternative sigma factors, sigma(E2) and sigma(E3), classified in the extracytoplasmic function sigma family for RNA polymerases, were identified in the deep-sea piezophilic bacterium Shewanella violacea DSS12. Amino acid alignments revealed that the domains for transcriptional functions were comparatively conserved compared with Escherichia coli sigma(E) in both proteins. Core-binding analysis suggested that both proteins function as sigma factors.

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier ((13)C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.

We have investigated the molecular phylogeny of cold-seep sediments obtained from the Nankai Trough, at depths of about 600, 2,000, and 3,300 m, and compared the microbial diversity profiles of those sediments samples. The gamma-Proteobacteria that might function as sulfide oxidizers and the symbiotically related delta-Proteobacteria which might function as sulfate reducers were identified amongst the bacteria from all depths of the sediments. However, anoxic methane oxidizing archaea (ANME) and methanogens were only found in the 600 m deep sediments. These results indicated that the cold-seep microbial sulfur circulation system could be functioning in the shallow seep sediment at a depth of 600 m and the microbial activities at these sites might be more dynamic than at other deeper cold-seep sites.

Deep-sea sediment samples were collected at a depth of 3,064 m in the Japan Sea. Microorganisms in the sediment sample were cultivated under several pressure conditions, and the high-pressure adapted microbes were isolated. Two of the isolates exhibited piezophilic growth profiles. This is the first report to show the presence of piezophiles in the Japan Sea.

Two genes for alternative sigma factors, sigma(E2) and sigma(E3), classified in the extracytoplasmic function sigma family for RNA polymerases, were identified in the deep-sea piezophilic bacterium Shewanella violacea DSS12. Amino acid alignments revealed that the domains for transcriptional functions were comparatively conserved compared with Escherichia coli sigma(E) in both proteins. Core-binding analysis suggested that both proteins function as sigma factors.

ABSTRACT: BACKGROUND: Dss1 (or Rpn15) is a recently identified subunit of the 26S proteasome regulatory particle. In addition to its function in the protein degradation machinery, it has been linked to BRCA2 (breast cancer susceptibility gene 2 product) and homologous DNA recombination, mRNA export, and exocytosis. While the fungal orthologues of Dss1 are not essential for viability, the significance of Dss1 in metazoans has remained unknown due to a lack of knockout animal models. RESULTS: In the current study deletion of dss-1 was studied in Caenorhabditis elegans with a dss-1 loss-of-function mutant and dss-1 directed RNAi. The analysis revealed an essential role for dss-1 in oogenesis. In addition, dss-1 RNAi caused embryonic lethality and larval arrest, presumably due to loss of the dss-1 mRNA maternal contribution. DSS-1::GFP fusion protein localised primarily in the nucleus. No apparent effect on proteasome function was found in dss-1 RNAi treated worms. However, expression of the C. elegans dss-1 in yeast cells deleted for its orthologue SEM1 rescued their temperature-sensitive growth phenotype, and partially rescued the accumulation of polyubiquitinated proteins in these cells. CONCLUSIONS: The first knockout animal model for the gene encoding the proteasome subunit DSS-1/Rpn15/Sem1 is characterised in this study. In contrast to unicellular eukaryotes, the C. elegans dss-1 encodes an essential protein, which is required for embryogenesis, larval growth, and oogenesis, and which is functionally conserved with its yeast and human homologues.

Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.

Electricity-generating bacteria have attracted intensive studies in recent years as an important resource for energy generation. In this investigation, a bacterial isolate capable of producing electric current during fermentation processes was obtained from a deep-sea hydrothermal field in the Atlantic Ocean for the first time. The strain, assigned to Shewanella sp. DS1 based on its 16S-rRNA sequence analysis, grew at the optimum temperature of 30 degrees C and optimum pH of 6.5. The results showed that the electric current generated by the strain increased during the first few hours and eventually reached the maximum (0.29 mA) at approximately 15 hours after inoculation. The electric current, however, decreased slowly as time increased. Thus, our study demonstrated that the deep sea promises to be a good reservoir for screening electricity-generating microbes.

Two strains belonging to the genus Shewanella, HAW-EB2(T) and HAW-EB5(T), were isolated previously from marine sediment sampled from the Atlantic Ocean, near Halifax harbour in Canada, for their potential to degrade explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In the present study, strains HAW-EB2(T) and HAW-EB5(T) were found to display high 16S rRNA gene sequence similarity (90-99.5 %) to species of Shewanella, but their gyrB sequences were significantly different from each other and from species of Shewanella (79-87.6 %). Furthermore, DNA-DNA hybridization showed that the genomic DNA of the two strains was only 22 % related and showed less than 41 % relatedness to closely related species of Shewanella. In comparison to other species of Shewanella, strains HAW-EB2(T) and HAW-EB5(T) were also unique in some phenotypic properties such as activities of beta-galactosidase and tyrosine arylamidase and the ability to metabolize certain organic acids and sugars. Both strains HAW-EB2(T) and HAW-EB5(T) utilize malate, valerate, peptone and yeast extract as sole carbon and energy sources. The major membrane fatty acids of the two strains were C(14 : 0), iso-C(15 : 0), C(16 : 0), C(16 : 1)omega7, C(18 : 1)omega7 and C(20 : 5)omega3 and their major quinones were Q-7, Q-8 and MK-7. On the basis of these results, strain HAW-EB2(T)(=NCIMB 14238(T)=CCUG 54553(T)) is proposed as the type strain of Shewanella canadensis sp. nov. and strain HAW-EB5(T)(=NCIMB 14239(T)=CCUG 54554(T)) is proposed as the type strain of Shewanella atlantica sp. nov.

A Gram-negative, motile, rod-shaped, psychrophilic bacterium, LT17(T), was isolated from deep-sea sediments (3300 m depth) of the East Sea (Sea of Japan). Optimal growth of LT17(T) requires the presence of 2.5 %(w/v) NaCl, a pH of 7.0-7.5 and a temperature of 17 degrees C. The isolate grows optimally under a hydrostatic pressure of 10 MPa and growth is possible between 0.1 and <30 MPa. The novel strain is positive in tests for catalase, oxidase, lipase, beta-glucosidase and gelatinase activities and reduces nitrate to nitrate. The predominant cellular fatty acids are iso-C13 : 0, iso-C15 : 0, C16 : 0, C16 : 1omega7 and C20 : 5omega3. The DNA G+C content of strain LT17(T) is 38.8 mol%. Phylogenetic analysis of 16S rRNA gene sequences places this bacterium in the class Gammaproteobacteria, within the genus Shewanella. The closest relatives of strain LT17(T) are Shewanella japonica (97.8 % gene sequence similarity), Shewanella pacifica (97.5 %), Shewanella olleyana (96.8 %), Shewanella frigidimarina (96.5 %) and Shewanella gelidimarina (95.4 %). The DNA-DNA hybridization levels between the novel isolate and its closest known phylogenetic relatives, S. japonica and S. pacifica, are lower than 14 %. On the basis of this polyphasic evidence, strain LT17(T) represents a novel species of the genus Shewanella, for which the name Shewanella donghaensis sp. nov. is proposed. The type strain is LT17(T)(=KCTC 10635BP(T)=JCM 12524(T)).

Two Shewanella-like bacterial strains, WP2(T) and WP3(T), which were isolated from west Pacific deep-sea sediment, were studied to determine their taxonomic position. Cells of the two bacteria were facultatively anaerobic, Gram-negative rods and motile by means of a single polar flagellum. Strain WP2(T) was psychrophilic, growing optimally at about 10-15 degrees C, whereas strain WP3(T) was psychrotolerant, growing optimally at 15-20 degrees C. The two strains grew in the pressure range 0.1-50 MPa, with optimal growth at 20 MPa. Strain WP3(T) was able to use nitrate, fumarate, trimethylamine N-oxide (TMAO), DMSO and insoluble Fe(III) as terminal electron acceptors during anaerobic growth, whereas strain WP2(T) was able to use only nitrate, TMAO and DMSO. The 16S rRNA gene sequences of strains WP2(T) and WP3(T) were 97 % identical, and showed highest similarity (97 %) to those of Shewanella fidelis KMM 3589 and Shewanella benthica ATCC 43992(T), respectively. The gyrB gene sequences of strains WP2(T)and WP3 (T) were also determined, and showed highest similarity to those of Shewanella violacea JCM 10179(T)(90 %) and Shewanella sairae SM2-1(T)(87 %), respectively. Contrary to the 16S rRNA gene sequence results, the phylogeny based on gyrB gene sequence analysis placed strain WP2(T), S. violacea and S. benthica in one group, while strain WP3(T) grouped with S. fidelis and S. sairae. DNA-DNA hybridization experiments supported the placement of strain WP2(T) with S. violacea and S. benthica. Phylogenetic evidence, together with DNA-DNA relatedness and phenotypic characteristics, indicated that the two new strains represented two novel deep-sea Shewanella species. The names Shewanella psychrophila sp. nov.(type strain WP2(T)=JCM 13876(T)=CGMCC 1.6159(T)) and Shewanella piezotolerans (type strain WP3(T)=JCM 13877(T)=CGMCC 1.6160(T)) are proposed.

Shewanella violacea DSS12 is a psychrophilic facultative piezophile isolated from the deep sea. In a previous study, we have shown that the bacterium adapted its respiratory components to alteration in growth pressure. This appears to be one of the bacterial adaptation mechanisms to high pressures. In this study, we measured the respiratory activities of S. violacea grown under various pressures. There was no significant difference between the cells grown under atmospheric pressure and a high pressure of 50 MPa relative to oxygen consumption of the cell-free extracts and inhibition patterns in the presence of KCN and antimycin A. Antimycin A did not inhibit the activity completely regardless of growth pressure, suggesting that there were complex III-containing and -eliminating pathways operating in parallel. On the other hand, there was a difference in the terminal oxidase activities. Our results showed that an inhibitor- and pressure-resistant terminal oxidase was expressed in the cells grown under high pressure. This property should contribute to the high-pressure adaptation mechanisms of S. violacea.

A psychrophilic bacterium, designated strain HJ039(T), was isolated from a marine sponge collected in the East Sea of Korea (also known as the Sea of Japan). Cells were Gram-negative, motile and rod-shaped (1.8-3.54 mumx0.27-0.73 mum). Growth was observed between 5 and 26 degrees C (optimum 15 degrees C), at pH 5.0-8.5 (optimum pH 6.0-6.5) and in the presence of 0-6.0 % NaCl (optimum 2.0 %). The 16S rRNA gene sequence of strain HJ039(T) showed high levels of similarity (93.7-95.4 %) with members of the genus Shewanella, especially with Shewanella gaetbuli TF-27(T)(95.2 %), Shewanella decolorationis S12(T)(94.9 %), Shewanella putrefaciens LMG 26268(T)(94.6 %), Shewanella hafniensis P010(T)(94.6 %), Shewanella algae ATCC 51192(T)(94.5 %) and Shewanella kaireitica c931(T)(94.5 %). However, phylogenetic analysis revealed that strain HJ039(T) shared a phyletic line with S. algae and Shewanella amazonensis. The major respiratory quinone was Q-8. The DNA G+C content was 52.8 mol%. The major fatty acids were i-13 : 0 (8.5 %), 15 : 0 (4.2 %), i-15 : 0 (23.2 %), i-15 : 1 (7.9 %), 16 : 0 (8.7 %), 16 : 1omega7 (21.0 %) and 17 : 1omega8 (6.4 %). From this polyphasic taxonomic evidence, strain HJ039(T) is considered to represent a novel species of the genus Shewanella, for which the name Shewanella spongiae sp. nov. is proposed. The type strain is HJ039(T)(=KCCM 42304(T)=JCM 13830(T)).