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Cytochrome c₅ of pressure-sensitive Shewanella livingstonensis (SL cytc₅) exhibits lower thermal stability than a highly homologous counterpart of pressure-tolerant Shewanella violacea. This stability difference is due to an enthalpic effect that can be attributed to the amino acid residue at position 50 (Leu or Lys). These cytc₅ proteins are appropriate materials for understanding the protein stability mechanism.

Pseudomonas sp. strain MT-1 was the first deep-sea denitrifier isolated and characterized from mud recovered from a depth of 11,000 m in the Mariana Trench. We report here the genome sequence of this bacterium, which contributes to our understanding of denitrification and bioenergetics in the deep sea.

When non-extremophiles encounter extreme environmental conditions, which are natural for the extremophiles, stress reactions, e.g., expression of heat shock proteins (HSPs), are thought to be induced for survival. To understand how the extremophiles live in such extreme environments, we studied the effects of high hydrostatic pressure on cellular contents of HSPs and their mRNAs during growth in a piezophilic bacterium, Shewanella violacea. HSPs increased at high hydrostatic pressures even when optimal for growth. The mRNAs and proteins of these HSPs significantly increased at higher hydrostatic pressure in S. violacea. In the non-piezophilic Escherichia coli, however, their mRNAs decreased, while their proteins did not change. Several transcriptional start sites (TSSs) for HSP genes were determined by the primer extension method and some of them showed hydrostatic pressure-dependent increase of the mRNAs. A major refolding target of one of the HSPs, chaperonin, at high hydrostatic pressure was shown to be RplB, a subunit of the 50S ribosome. These results suggested that in S. violacea, HSPs play essential roles, e.g., maintaining protein complex machinery including ribosomes, in the growth and viability at high hydrostatic pressure, and that, in their expression, the transcription is under the control of σ(32).

Hematopoietic stem cell transplantation (HSCT) is one curable option for high-risk acute lymphoblastic leukemia (ALL); however, transplant-related toxicities might be severe in patients with Down syndrome and ALL (DS-ALL).HSCTs performed in patients with DS-ALL were identified in the Japan Society for Hematopoietic Cell Transplantation registry.In the registry data, 11 patients with DS-ALL were identified. The median age at HSCT was 9 years (range: 6-22 years). Six patients underwent HSCT at non-remission status. Allogeneic grafts were utilized in all patients, including eight patients who received HSCT from unrelated donors. Reduced intensity conditioning regimens were used in three patients. All patients achieved neutrophil engraftment by a median of day 18 (range: day 11-61). Ten patients experienced grade 3 or more infectious episodes. Six patients experienced complications of the respiratory system. The incidences of II-IV or III-IV acute GVHD were nine (81.8%) or seven patients (63.6%), respectively. Chronic GVHD was observed in five (55.6%) out of nine evaluable patients. Seven patients died at a median of 6 months (range: 0-24 months) after HSCT. Two-year relapse-free and overall survival were 33.3% (95% CI: 2.5-64.1%) or 37.5% (95% CI: 5.9-69.1%), respectively. The causes of death were relapse (n = 2), infection (n = 2), bleeding (n = 1), thrombotic microangiopathy (n = 1), and chronic GVHD (n = 1).Therapy-related mortality accounted for five out of seven deceased patients in this case series. Attempts to reduce toxicities should be considered in HSCT for patients with DS-ALL.

This chapter focuses on the isolation, taxonomy, and diversity of piezophilic microorganisms and their habitats. Based upon several studies the authors have indicated that cultivated psychrophilic and piezophilic deep-sea bacteria could be affiliated with one of five genera within the Gammaproteobacteria subgroup: Shewanella, Photobacterium, Colwellia, Moritella, and Psychromonas, which was formally classified as ‘’an unidentified genus’’. The chapter describes taxonomic features of the piezophilic genera. For handling piezophiles for further study, JAMSTEC developed a deep-sea baropiezophile and thermophile isolation and cultivation system, referred to as the DEEPBATH system. The DEEPBATH system consists of four separate devices: (1) a pressure-retaining sampling device, (2) a dilution device under pressure conditions, (3) an isolation device, and (4) a cultivation device. From the analyses of 16S rRNA gene sequences after cultivation at 65 MPa, two groups of the bacterial genera Shewanella and Moritella were identified. The authors have analyzed the microbial community structures by the terminal restriction fragment length polymorphism for the bacterial 16S rRNA gene and determined that the community is drastically changed at different pressure conditions of cultivation using the DEEPBATH system. Piezophiles are characterized by high levels of unsaturated fatty acids in their cell membrane layers, but long-chain polyunsaturated fatty acid (PUFA) like EPA and DHA are not necessarily required for high-pressure growth. The diversity of piezophilic bacteria is closely linked with the global deep-sea ocean circulation, but some of the closed oceans, like the Japan Sea, also contain piezophilic bacteria taxonomically similar to deep-sea microbes in the open oceans.

This chapter focuses on the molecular characteristics of pressure adaptation in Shewanella violacea DSS12 and recent advances in developing genetics and utilizing genomics. Sigma 54 plays an important role in pressure-regulated transcription in S. violacea, although it should be noted that the expression of sigma 54 is not itself regulated by pressure. Any of these trans-acting factors (sigma 54, NtrC, or NtrB) could play an important role in pressure-regulated transcription in this piezophilic bacterium. Downstream from the pressure-regulated operon described is an open reading frame (ORF) homologous to the cydD gene of E. coli. Another aspect of transcription in piezophiles is the stability of the quaternary structure of RNA polymerase. It is likely that the S. violacea sigma subunit stabilizes the core enzyme through alteration of the quaternary structure of RNA polymerase, resulting in piezotolerance. One possible explanation for the low conjugation frequency is that the optimal mating temperature for DSS12R is 20°C, which is much lower than the optimal temperature for E. coli. This study provided the first demonstration of gene transfer in the piezophilic bacterium DSS12. The study of the mechanisms for adaptation to high-pressure environments, including gene regulatory systems, may now also proceed in vivo using genetic approaches in this piezophile. Therefore, genomic analysis of marine extremophiles may lead to the discovery of new functions for genes.

This chapter focuses on studies of the microbial diversity in deep-sea methane-impacted sediments using culture-independent procedures. It discusses sulfur and carbon cycling ecosystems in chemosynthetic pathways which are independent of solar-power-dependent energy-generating systems. Microbial diversity studies in deep-sea sediments have been performed at depths ranging from ~1,000 m, in Sagami Bay, to ~11,000 m, in the Mariana Trench Challenger Deep. To study the different cold-seep microbial ecosystems present between the Japan Trench land slope and the Nankai Trough, the microbial diversity of Nankai Trough cold-seep sites at different depths was investigated and correlations were sought between the microbial communities and their geological settings. Methylotrophs are a group of bacteria which can utilize methane (methanotrophs) and/or a variety of other one-carbon (C1) compounds more reduced than formic acid, such as methanol and methylated amines, as sole carbon and energy sources. The levels of methylotrophs in the sediments of the tropical West Pacific Warm Pool (WP) were semiquantified by quantitative competitive PCR. It was found that the WP contained around 3 * 104 to 3 * 105 molecules of mxaF gene copy per gram of sediment. Using this method, the distribution and abundance of methylotrophs in deep-sea sediments from the West Pacific WP were compared with those in east and middle Pacific deep-sea sediments, seashore sediments, and flower garden and rice field soils.

The term ‘deep ocean’ typically describes any marine ecosystem located at depths higher than 500 m. This environment is characterised by an elevated hydrostatic pressure, an average temperature of 2–4 °C, the absence of sunlight and the scarce availability of organic food. Specific organisms are associated with the deepest areas, and pressure-adapted animals as well as microorganisms inhabit these peculiar ecosystems. It is difficult to understand how we do not know much about deep sea compared to space environment, despite the fact that its distance from the ocean surface is relatively short. With no doubt, the deep-sea biodiversity is still mostly unseen and important benefits to human health and industrial activities will be available in the future from more extensive studies on this fascinating environment. Key Concepts: The deep sea is the world's largest ecosystem. The deep-sea biodiversity is still mostly unseen. The less diversity of animals with increasing depths is mainly due to the hydrostatic pressure, as well as to a larger competition for food coupled with a lower basal metabolism. Further insights into the natural traits of deep-sea will lead to important benefits to human health and industrial applications. Despite the fact that the distance of deep-sea from the ocean surface is much shorter than that between Earth and space, the abyssal environment is far to be well known. Keywords: deep-sea ecosystems; biodiversity; taxonomy; biotechnology; technologies

Shewanella species live widely in deep-sea and shallow-water areas, and thus grow piezophilically and piezosensitively. Piezophilic and psychrophilic Shewanella benthica cytochrome c 5 (SB cytc 5) was the most stable against guanidine hydrochloride (GdnHCl) and thermal denaturation, followed by less piezophilic but still psychrophilic Shewanella violacea cytochrome c 5 (SV cytc 5). These two were followed, as to stability level, by piezosensitive and mesophilic Shewanella amazonensis cytochrome c 5 (SA cytc 5), and piezosensitive and psychrophilic Shewanella livingstonensis cytochrome c 5 (SL cytc 5). The midpoint GdnHCl concentrations of SB cytc 5, SV cytc 5, SL cytc 5, and SA cytc 5 correlated with the optimal growth pressures of the species, the correlation coefficient value being 0.93. A similar trend was observed for thermal denaturation. Therefore, the stability of each cytochrome c 5 is related directly to its host's optimal growth pressure. Phylogenetic analysis indicated that Lys-37, Ala-41, and Leu-50 conserved in piezosensitive SL cytc 5 and SA cytc 5 are ancestors of the corresponding residues in piezophilic SB cytc 5 and SV cytc 5, Gln, Thr, and Lys, respectively, which might have been introduced during evolution on adaption to environmental pressure. The monomeric Shewanella cytochromes c 5 are suitable tools for examining protein stability with regard to the optimal growth pressures of the source species.

We investigated whether vitamin E (VE) supplementation alleviates collagen-induced arthritis in its stages by altering immune functions in DBA/1J mice. Mice were divided into 4 groups; control (VE 30 IU/kg in diet), A-VE0, A-VE30 and A-VE500 (animals with induced arthritis given VE 0, VE 30, VE 500 IU/kg in diet respectively). Arthritis was induced with Type II Collagen (TIIC). The extent of arthritis in the joint did not decline in neither A-VE0 nor A-VE500 groups compared with A-VE30. The proliferation of splenocytes stimulated with ConA was suppressed in both A-VE0 and A-VE500 groups. The production of Th1 cytokine stimulated with TIIC was increased in both A-VE0 and A-VE500 groups, but production of Th2 cytokine was not changed. Our observations suggest that VE supplementation does not always improve the progress of arthritis. The influence of VE on cytokine balance in autoimmune (Th1 predominant) arthritis should be further investigated.

Five passenger vehicles used around the Detroit area were submitted for corrosion investigation. They were manufactured in 1989 and 1991. Panels forming the body shell were classified into five categories according to the corrosion environment, and their cosmetic corrosion and perforation corrosion were evaluated. It was confirmed that exterior panels made of two-sided zinc coated steel sheet has excellent performance in cosmetic corrosion, and it was shown that the corrosion depth at lapped parts under low humid conditions decreases with increased zinc coating weight. For precoated panels with zinc coating more than 45 g/m 2 , the degree of corrosion was very low.

The bacterial endosymbionts of the thyasirid clam Maorithyas hadalis collected from the hadal zone in the Japan Trench were characterized. Two distinct phylotypes of endosymbiotic bacte- ria were discovered within the gill tissues by molecular phylogenetic analysis and in situ hybridiza- tion. Symbiont Type I was affiliated with thioautotrophic symbionts of vesicomyid clams and deep-sea mussels from deep-sea hydrothermal vents and cold seeps. Symbiont Type II was not related to pre- viously reported bacterial symbionts, and was distantly related to the free-living chemoautotrophic bacteria (genera Thiomicrospira and Hydrogenovibrio). In situ hybridization experiments indicated spatial partitioning between the 2 M. hadalis symbionts, with Symbiont I occurring mainly in the outer regions of bacteriocyte zones and Symbiont II situated predominantly within inner regions of bacteriocyte zones. This is the deepest chemosynthetic symbiosis ever recorded and the first report to show spatial partitioning between the intracellular endosymbionts in marine invertebrates.

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.

9 páginas.-- 3 figuras.-- referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, A new anaerobic, thermophilic, CO-utilizing marine bacterium, strain JMT, was isolated from a submarine hot vent in Okinawa Trough. Cells of strain JMT were non-motile thin straight rods, sometimes branching, with a cell wall of the Gram-positive type, surrounded with an S-layer. Chains of three to five cells were often observed. The isolate grew chemolithotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O-->CO2+H2) and organotrophically on peptone, yeast extract, starch, cellobiose, glucose, galactose, fructose and pyruvate, producing H2, acetate and CO2. Growth was observed from 50 to 80 degrees C with an optimum at 70 degrees C. The optimum pH was 6.8-7.1. The optimum concentration of sea salts in the medium was 20.5-25.5 g l(-1). The generation time under optimal conditions was 7.1 h. The DNA G+C content was 33 mol %. Growth of isolate JMT was not inhibited by penicillin, but ampicillin, streptomycin, kanamycin and neomycin completely inhibited growth. The results of 16S rDNA sequence analysis revealed that strain JMT belongs to the Thermoanaerobacter phylogenetic group within the Bacillus-Clostridium subphylum of Gram-positive bacteria but represents a separate branch of this group. On the basis of morphological and physiological features and phylogenetic data, this isolate should be assigned to a new genus, for which the name Carboxydobrachium is proposed. The type species is Carboxydobrachium pacificum; the type strain is JMT (= DSM 12653T)., The authors are grateful to G. A. Zavarzin and A. V. Lebedinsky, Institute of Microbiology, Russian Academy of Sciences, for helpful discussions. This work was supported by the Russian Foundation for Basic Research, grants nos 96-04-49463 and 99-04-48360, the ‘Biodiversity’ program of the Russian Ministry of Science and Technology, a NATO CLG grant to T. S. and F. T. R. and by NSF LEXEN Program support to F. T. R. and J. M. G.

栄養士養成課程に学ぶ若年女性における日常の昼食摂取状況の問題点を明らかにするとともに, 給食管理実習の役割について考察することを目的に, 栄養所要量の3/8量の基準量からなる給食を10回にわたって提供し, アンケートによる給食の残菜調査と3日間の食物摂取状況調査及び消費エネルギー量調査, 日常の食習慣に関するアンケート調査を実施した。その結果, 以下の知見が得られた。1) BMIを基に体型分類をした結果,“やせ”と判定される者は14.9%,“普通”が83.0%,“肥満”が2.1%であった。2) 日常の摂取エネルギー量は, 消費エネルギーの推定値に対して, 平均で約82%であった。3) 日常の昼食の栄養素等摂取量は, 栄養所要量の3/8量と比較して, 49.3～76.9%の範囲で, 食事内容は“主菜のない昼食”が多かった (74.4%)。4) “献立”,“主食”,“主菜”,“副菜”別に給食を全て食べた者の割合は, それぞれ平均値で65.8%, 73.5%, 87.2%, 84.4%であった。5) 和風・洋風・中華風といった献立様式と, 提供栄養量や残菜の有無, 味との間に有意な関連性はみられなかった。6) 給食を残す理由は,“主食”では分量が多いため,“副菜”ではまずいためと答えていた。7) 朝食をとらなかった者に給食を残す割合が高かった。以上のことから, 給食管理実習は, 一定基準の栄養量を提供する方法を学ぶ場であるだけでなく, 適正な栄養素摂取量となる献立を体得させる食教育の場としての役割がある。

A glutamine synthetase gene ( glnA ) was isolated from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12. A 7.5-kb Sac I fragment containing the complete glnA gene was cloned and sequenced. The glnA gene was found to encode a protein consisting of 469 amino acid residues, showing 75.0% identity to the glutamine synthetase of Escherichia coli . Primer extension analyses revealed two transcription initiation sites in glnA and expression from each site was positively regulated by pressure. Putative promoters recognized by σ 70 and σ 54 were identified in the region upstream of glnA . An electrophoretic mobility shift assay demonstrated that S. violacea σ 54 specifically binds to the promoter region of glnA , suggesting that σ 54 may play an important role in pressure-regulated transcription in this piezophilic bacterium.

Although decreased T-cell function has been observed in obese human subjects and genetically obese animals, the precise role of immune functions in obesity is still unclear. To investigate immune functions in obesity, we examined the proliferative responses of splenic lymphocytes and their capacity to produce cytokines in the presence or absence of leptin, the protein produced by the obese gene, in diet-induced obese and control mice. For induction of obesity, C57BL/6J mice were fed a high-fat diet for 13 weeks. In mice fed the high-fat diet, body weight, fat pad weight, and tumor necrosis factor (TNF) alpha production by adipocytes were significantly increased relative to mice fed the normal diet. Lipopolysaccharide (LPS) stimulated proliferation of cultured splenocytes from diet-induced obese mice was also increased. However, production of interleukin (IL)-2 by splenic lymphocytes from obese mice was suppressed, whereas interferon (IFN)-gamma and IL-4 production was increased. Exogenous lepitn regulated the cytokine production by cultured splenocytes from control and obese mice, respectively (upregulation of IFN-gamma and downregulation of IL-2 in control mice, and downregulation of IL-4 in obese mice). These results suggest that changes in cytokine production by splenic lymphocytes in obesity are indicative of altered immune functions that might contribute to related complications, although the effect of difference in nutrient intake (macro and micro) may also have contributed to the changes.

The membrane phospholipids of two barophilic bacteria, DB21MT-2 and DB21MT-5, isolated from sediments from the Marianas Trench at 11,000 m were structurally characterized and quantitatively determined by liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS). Phospholipids detected in the barophiles were distributed in five different classes: phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), diphosphatidylglycerol (DPG), and phosphatidylethanolamine (PE) and its methylated forms phosphatidylmethylethanolamine (PME) and phosphatidyldimethylethanolamine (PDME). Concentrations of phospholipids/phospholipid groups range from 5.3 to 4.583 μg/g (dry weight). A unique aspect of the phospholipid profiles of the barophilic bacteria was the wide distribution of polyunsaturated fatty acids 20 : 5 (in DB21MT-2) and 22 : 6 (in both DB21MT-5 and DB1MT-2) on the sn -1 and mostly on the sn -2 position of the phospholipids. The results suggest that the adaptation of the barophiles to low temperature and high hydrostatic pressure influenced the synthesis of phospholipids containing polyunsaturated fatty acids. It was also observed that the polyunsaturated fatty acids were associated with almost every phosphatidylglycerol (PG) molecule. Presumably, the larger head group of PG would be expected to cause greater disruption in acyl chain packing within the membrane bilayer and thereby lower the transition temperature in response to the additive effects of low temperature and high pressure. The detection of phospholipids with polyunsaturated fatty acids also has important geochemical implications for paleoenvironment reconstruction and for determining the surface water biological productivity of the ocean. It seems that psychrophilic and barophilic bacteria may be major sources of polyunsaturated fatty acids to the deep-sea sediments, given the fact that the vertical flux of polyunsaturated fatty acids from surface water plankton decreased rapidly with depth.

Azacycloundecenes, which are key intermediates in the synthesis of derivatives of the marine alkaloid manzamine C, are conveniently prepared using ring-closing olefin metathesis (RCM).

3-isopropylmalate dehydrogenase (IPMDH)-encoding leuB genes were obtained from the obligate piezophile Shewanella benthica DB21MT-2 and non-piezophile Shewanella oneidensis MR-1. The genes were expressed in Escherichia coli and the proteins were purified using His-tag. The estimated kinetic parameters of these enzymes indicated that IPMDH of S. benthica DB21MT-2 is more tolerant of high pressure than that of S. oneidensis MR-1. Thus such an adaptation is one of the mechanisms bacteria utilize for survival at high pressures.

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.

Potential regulatory elements were identified upstream of the pressure-regulated operon in the deep-sea barophilic bacterium Shewanella violacea strain DSS12 and binding of cellular proteins to this region was studied under different pressure conditions. Sequence analysis revealed the existence of a region, designated region A, showing similarity to the consensus sequence for σ54 binding, when the region upstream of the operon was compared with several consensus sequences for σ factors. A palindromic sequence AGTTAAAGATTAAACT, designated region B, was found further upstream beyond region A. In a region designated region C, just upstream of region B, a unique octamer motif AAGGTAAG, tandemly repeated 13 times, was found. By means of an electrophoretic mobility shift assay it was demonstrated that a σ54-like factor recognized region A while other unknown factors recognized the sequences in regions B and C. Different shift patterns of protein-DNA complexes were observed when extracts of cells cultured at 0.1 MPa and 50 MPa were incubated with 32P-labeled DNA probes corresponding to region B or C. These results indicate that barophilic strain DSS12 expresses different DNA-binding factors capable of recognizing these elements upstream of the pressure-regulated operon under different pressure conditions.

Dense populations of bivalves, primarily Calyptogena sp., were observed at cold seeps of the Nankai Trough. Bacterial input to the sediment was estimated through determination of phospholipid ester-linked fatty acid (PLFA) and DNA profiles. Results indicated a bacterial biomass of 109 cells (g dry wt)-1 while individual fatty acid profiles revealed a predominance of monounsaturated fatty acids, mainly 18:1 isomers. The presence of these fatty acids can be interpreted to reflect a response to low temperature and a predominance of psychrophilic bacteria. DNA fragments encoding bacterial ribosomal RNA small-subunit sequences (16S rDNA) were amplified by the polymerase chain reaction method using DNA extracted directly from the sediment samples. From the sequencing results, at least 19 kinds of bacterial 16S rDNAs related to mostly the Proteobacteria and a few gram-positive bacteria were identified. These results suggest that the bacterial community in the Nankai Trough sediments consists of mainly bacteria belonging to the Proteobacteria γ, e, and δ subdivisions. Bacteria belonging to the e and δ subdivisions, which are known to include epibiont and sulfate reducing bacteria, respectively, were mostly detected in the sediment obtained from inside the area of the Calyptogena community, and the δ-Proteobacteria may function to supply reduced sulfur to bacterial endosymbionts of Calyptogena.

Seven sediment samples have been examined, taken from different depths of the deep-sea in the range of 1159m to 6482m. A total of 75 different 16S rDNA sequences (149 clones) analyzed clustered into the Proteobacteria, Gram-positive bacteria, Cytophaga, Planctomyces, and Actinomycetes and many sequences were from microorganisms that showed no phylogenetic affiliation with known bacteria. Clones identical to 16S rDNA sequences of members of the genus Pseudomonas were observed in all of the sediments examined. The second group of common sequences cloned from six sediment samples was related to the 16S rDNA sequence of a chemoautotrophic bacterium, the Solemya velum symbiont. Five 16S rDNA sequences from three sediments were related to those of the Alvinella pompejana epibiont which is a member of the e-Proteobacteria. Only one sequence was obtained that was closely related to the 16S rDNA of the barophilic bacterium, Shewanella benthica, which might be a minor population in the deeper sediments. δ-Proteobacteria-related sequences were cloned from sediments obtained from sites near man-made garbage deposits and a Calyptogena community. These environments obviously would be richer in nutrients than other sites, and might be expected to show more types of bacteria than other deep-sea sediments. A large number of cloned sequences in this study showed very low identity to known sequences. These sequences may represent communities of as-yet-uncultivated microorganisms in the sediments.

A sample of deep-sea sediment was obtained from the Japan Trench at a depth of 6292 m using a pressure-retaining sediment sampler. Microorganisms in the sediment sample were cultivated in marine broth 2216 at ambient pressure (65 MPa) without decompression, and at atmospheric pressure (0.1 MPa) as a control experiment. 16S ribosomal RNA genes (rDNA) were amplified by PCR from DNA extracted from the original sediment sample and the mixed cultures, and the nucleotide sequences were determined. The results of phylogenetic analysis based on 16S rDNA sequences indicated that microbial diversity in the original sediment samples showed a wide distribution of types in the domain Bacteria. Furthermore, in the mixed cultures incubated at 65 MPa without decompression, bacterial strains belonging to the Shewanella barophiles branch and the genus Moritella existed together at the beginning of cultivation, and Moritella strains became dominant towards the end of the cultivation period. Finally, in the mixed cultures incubated at atmospheric pressure, strains belonging to the genus Pseudomonas were dominant at all times. Analysis of fatty acids extracted from the cultures supported the phylogenetic results.

Several baro (piezo) -philic microorganisms have been isolated from a deep-sea high hydrostatic pressure environment in our laboratory. The results of taxonomic studies showed that all of the barophile isolates belonged to Proteobacteria gamma-subgroup, and four novel species were identified. The moderately barophilic bacterium, Shewanella violacea strain DSS 12 was one of them, and this strain was able to grow from atmospheric pressure (0. 1 MPa) to 70 MPa conditions in the same way. Molecular mechanisms of pressure-regulation on gene expression in S. violacea were analyzed, and we identified particular DNA binding proteins that might be essentially important for pressure regulation. Atmospheric pressure adapted Escherichia coli that belonged to the same gamma-subgroup of the isolated barophilic species was also studied and we could observe the positive and negative effects on pressure regulation. Finally, we observed that hyper-thermophilic archaea could grow well under higher pressure conditions at higher temperatures. These results indicated that pressure could affect microorganisms' survival in several ways in such an environment.

Deep-sea soft sediments from trench systems and depths in the northwestern Pacific Ocean ranging from less than 300 to 10,897 m in depth have been analyzed for three target genera of actinomycetes: Micromonospora, Rhodococcus, and Streptomyces. Only culturable strains, recovered at atmospheric pressure on selective isolation media, have been examined to date. Maximum recoveries of culturable bacteria were greater that 10(7)/ml wet g sediment, but actinomycetes comprised a small proportion of this population (usually less than 1%). The target actinomycetes were isolated at all depths except from the Mariana Trench sediments. Actinomycete colonies were defined initially on the basis of colony morphologies, and preliminary identification then was made by chemotaxonomic tests. Pyrolysis mass spectrometry (PyMS) of deep-sea mycolic acid-containing actinomycetes gave excellent correspondence with numerical (phenetic) taxonomic analyses and subsequently was adopted as a rapid procedure for assessing taxonomic diversity. PyMS analysis enabled several clusters of deep-sea rhodococci to be distinguished that are quite distinct from all type strains. 16S rRNA gene sequence analysis has revealed that several of these marine rhodococci have sequences that are very similar to certain terrestrial species of Rhodococcus and to Dietzia. There is evidence for the intrusion of terrestrial runoff into these deep trench systems, and the inconsistency of the phenotypic and molecular taxonomies may reflect recent speciatiion events in actinomycetes under the high-pressure conditions of the deep sea. The results of DNA-DNA pairing experiments point to the novelty of Rhodococcus strains recovered from hadal depths in the Izu Bonin Trench. Biotransformation studies of deep-sea bacteria have focused on nitrile compounds. Nitrile-metabolizing bacteria, closely related to rhodococci, have been isolated that grow well at low temperature, high salt concentrations, and high pressures, suggesting that they are of marine origin or have adapted to the deep-sea environment.

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15kDa, and it contained 0.96 mol of protoheme and 1.95mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN-, and the catalytic activity of the enzyme was 50% inhibited by 4 microM CN-. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F.

From a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F, a membrane-bound cytochrome c-551 and a cytoplasmic cytochrome c-552 were purified. The cytochrome c-551 contained 44.2 nmol of heme c mg protein−1 and cytochrome c-552 contained 31.3 nmol of heme c mg protein−1. The CO difference spectrum of cytochrome c-551 showed a peak at 413.7 nm and troughs at 423.2, 522 and 552 nm which indicated that this cytochrome combined with CO. Cytochrome c-551 was found to consist of two subunits with molecular masses of 29.1 kDa and 14.7 kDa, respectively, and each subunit contained one heme c molecule. Cytochrome c-552 also consisted of two subunits with molecular masses of 16.9 kDa and 14.7 kDa, respectively, and only one of these subunits contained heme c. Cytochrome c-551 was constitutively synthesized when the cells were grown at pressures of either 0.1 MPa or 60 MPa, whereas cytochrome c-552 was synthesized only at 0.1 MPa. These results together with the results of analysis of membrane-associated catalytic activities suggest that the respiratory system of DB-172F is regulated by pressure and may be intimately related to the baroadaptability mechanism of this deep-sea bacterium.

Two strains of obligately barophilic bacteria were isolated from a sample of the world’s deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella . The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment.

DNA regions corresponding to portions of two different pressure-regulated operons previously identified in two deep-sea barophilic bacteria were separately PCR amplified from a variety of deep-sea microorganisms and sequenced. With the two sets of primers employed, amplification was particularly successful from the more barophilic bacteria examined. 16S rRNA sequence analysis revealed that these bacteria are all phylogenetically related and belong in a sub-branch of the genus Shewanella containing only the deep-sea Shewanella barophilic bacteria. We define this sub-branch as the ‘Shewanella barophile branch’ containing at least two different species. Our results suggest that the DNA sequences of the pressureregulated operons can be regarded as marker sequences to identify the Shewanella barophilic strains. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30 degrees C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4 degrees C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies.

Recently, so many genome analysis programs of the microorganisms have been investigating in the world, and some of them (5 strains) have finished and opened the sequencing data on computer net-work. From these results, we realyzed that the genome size of microorganisms and the quantity of regulatory functional genes and pseudo genes were corresponding. For example, 134 regulatory genes were identified in Synechocystis sp. PCC 6803 (3.6 Mb), however only 7 regulatory genes in Methanococcus Jannaschii (1.7 Mb). We believe that such difference of the genome size is corresponded to the quantity of the environmental stresses. Thus, this is very important to study the smaller genome organisms like hyperthermophilic archaea to realize the minimum genome for organisms. In my country, the genome project of new hyperthermophilic archaeon, Pyrococcus horikoshii, is in progress. P. horikoshii showed obligately barophilic growth profile at maximum high temperature (103°C), thus we may expect that the barophily of the deep-sea hyperthermophiles will be more understandable to consider about the origin of life and its evolution from the its genomic results.

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 facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.

Hydrostatic pressure analysis is an ideal approach for studying protein dynamics and hydration. The development of full ocean depth submersibles and high pressure biological techniques allows us to investigate enzymes from deep-sea organisms at the molecular level. The aim of this review was to overview the thermodynamic and functional characteristics of deep-sea enzymes as revealed by pressure axis analysis after giving a brief introduction to the thermodynamic principles underlying the effects of pressure on the structural stability and function of enzymes.

We have cloned the gene encoding RNA polymerase alpha subunit from a gene library of deep-sea barophilic bacterium strain DB6705. The clone contains the genes for ribosomal protein S4, RNA polymerase subunit alpha and ribosomal protein L17 in this order. The alpha gene has 328 amino acids and a molecular mass of 36 100 Da with 86.9% identity to Escherichia coli alpha gene. Differences between the two sequences were mainly in the N-terminal portion of the alpha subunit, which is involved in the assembly of the core RNA polymerase; while the 87 C-terminal residues, which form a region involved in contact with some positive regulators and rrnB P1 promoter region called UP-element, were identical in the both strain. Plasmid encoding the alpha subunit with an N-terminal hexahistidine tag was constructed. Using the plasmid, the recombinant fusion alpha subunit was overexpressed and successfully purified to near homogeneity.

The formation of plaques by lambda phage in Escherichia coli was prevented by elevated hydrostatic pressure; phage plaques were not detected at 30 MPa. Furthermore, using promoter fragments derived from the malB operon, we showed that gene expression initiated from both promoters (malK-lamB and malEFG) was repressed by elevated hydrostatic pressure. Our findings suggest that high pressure affects gene expression directed by the malB regulatory interval, and this may cause a decrease in the quantities of lambda receptor protein, LamB.

Fuel reprocessing plant is important for atomic energy cycle which is aimed to re-use uranium (U) and plutonium (Pu) from the spent nuclear fuels after barning-out in light water reactor. PUREX process, in which boiling nitric acid is used for dissolving fuel elements, is adopted in Japan. Many structural materials are used stainless steels protected by passive layers of the surface. At the beginning of developing the plant, many troubles of intergranular corrosion of stainless steels were experienced. According to the investigations of these problems, very low carbon type austenitic stainless steels were utilized for the materials. However, these stainless steels were corroded in some cases. For example, addition of hexavalent chromium (Cr (VI)) is heavily accelerated corrosion rate of these stainless steels in boiling nitric acid solutions. The corrosion potential of stainless steel in Cr (VI) added solution is much higher than that in pure nitric acid solution. The authors introduced that heptavalent vanadium (V (V)) was also accelerated corrosion rate of these stainless steels but the tendency of acceleration process was different from the case of containing Cr (VI). We analyzed the valence stats of Cr and V during corrosion test and showed that Cr (VI) reduced to Cr (III) with corrosion of stainless steel propagating. On the other hand, amount of V (V) was not changed during the corrosion test. It was concluded that V (IV) was easily re-oxidized in boiling nitric acid solution. Namely, V (V) is reduced to V (IV) on stainless steel surface by cathodic reaction of corrosion and then V (IV) is re-oxidized immediately to V (V) in the balk nitric acid solution. These data were obtained using non-radioactive solutions in the ordinary laboratory. The present work is aimed to compare the data of re-oxidation rate of actinides which are included in the actual solutions used in a reprocessing plant. We implemented a specially designed electrochemical test cell used for analyzing the valence states of actinide cations, i.e. neptunium (Np) and Pu, in a boiling nitric acid solution. The cell was set into glovebox in a hot laboratory which could be handled nuclear fuel materials. The cell was designed for minimizing the solution. The amounts of solution used are about 15 cm3 and sample size of working electrode is about 4 mm2. Electrochemical measurements and spectrum analyses were conducted with this apparatus. Analyzed cations were Cr (VI), V (V), Pu (VI) and Np (VI). Polarization curves showed that corrosion potentials of stainless steels changed to higher direction by addition of these cations and also corrosion current densities increased. The higher order of those is Cr (VI), Np (VI), V (V) and Pu (VI). Re-oxidation rates were obtained by the changes of absorption spectra with time using lower valence state ions added solutions, i.e. Cr (III), V (IV), Pu (IV) and Np (V). These species are re-oxidized to Cr (VI), V (V), Pu (VI) and Np (VI), respectively. Re-oxidation rates of them were very much different among them. Very fast re-oxidation rates were obtained by the V (IV) and Np (V) added solutions. A very low re-oxidation rate was obtained by the Cr (III) added solution. The Pu (IV) added solution was in the middle of them. The corrosion of stainless steel is controlled by corrosion potential of cation added solution at the initial stage and controlled by re-oxidation rate of cation as time passed. The element Np which indicates high corrosion potential and has fast re-oxidation rate influences too much the corrosion of stainless steel in boiling nitic acid solution among these elements in consideration.

Several barophilic and barotolerant bacteria were isolated from deep-sea mud samples of Suruga Bay (2485 m depth), the Ryukyu Trench (5110 m depth), and the Japan Trench (land-side 6356 m, and sea-side 6269 m depth, respectivelys. The barophilic bacteria, strains DB5501, DB6101, DB6705 and DB6906, were albe to grow better under high hydrostatic pressures than under atmospheric pressure (0.1 megapascals; MPa). The optimal growth pressures for the barophilic bacteria were approximately 50 MPa at 10°C. The barotolerant strains DSK1 and DSS12 were determined to be psychrophilic, and had optimal growth temperatures of 10°C and 8°C, respectively. The degree of barophily and barotolerance was shown to be very dependent on temperature. For example, at 4°C the barophilic strains were indistinguishable from barotolerant bacteria, whereas at 15°C the barotolerant strains behaved more like the barophilic strains. Based on sequence analysis of 16S ribosomal DNA, all of the strains included in this study belong to the gamma subgroup of the Proteobacteria. Phylogenetic relations between the isolated strains and the known gamma subgroup bacteria suggested that the isolated strains belong to a new sub-branch of this group.