Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions.

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Methylocaldum marinum sp. nov., a thermotolerant, methane-oxidizing bacterium isolated from marine sediments, and emended description of the genus Methylocaldum.

An aerobic, methane-oxidizing bacterium (strain S8(T)) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain is closely related to members of the genus Methylocaldum (97.6-97.9 % similarity) within the class Gammaproteobacteria. Strain S8(T) was a Gram-staining-negative, non-motile, coccoid or short rod-shaped organism. The temperature range for growth of strain S8(T) was 20-47 °C (optimum growth at 36 °C). It required NaCl (>0.5 %), tolerated up to 5 % NaCl and utilized methane and methanol. The major cellular fatty acid and major respiratory quinone were C16 : 0 and 18-methylene ubiquinone 8, respectively. The DNA G+C content was 59.7 mol%. Strain S8(T) possessed mmoX, which encodes soluble methane monooxygenase, as well as pmoA, which encodes the particulate methane monooxygenase. On the basis of this morphological, physiological, biochemical and genetic information, the first marine species in the genus Methylocaldum is proposed, with the name Methylocaldum marinum sp. nov. The type strain is S8(T) ( = NBRC 109686(T) = DSM 27392(T)). An emended description of the genus Methylocaldum is also provided.

Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic bacterium isolated from marine sediments near a hydrothermal vent.

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4(T)) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4(T) was closely related to members of the genus 'Methyloligella' (94.7% similarity) within the class Alphaproteobacteria. Strain Gela4(T) was a Gram-staining-negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4(T) was 19-43 °C (optimal growth at 35 °C). Strain Gela4(T) tolerated up to 9% NaCl with an optimum at 1%. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4(T) ( = NBRC 109540(T) = DSM 27242(T)) as the type strain.

Biogeography and biodiversity in sulfide structures of active and inactive vents at deep-sea hydrothermal fields of the Southern Mariana Trough.

The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and microbial ecosystems in marine environments.

Abundance of Zetaproteobacteria within crustal fluids in back-arc hydrothermal fields of the Southern Mariana Trough.

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.

Thermococcus coalescens sp. nov., a cell-fusing hyperthermophilic archaeon from Suiyo Seamount.

A cell-fusing hyperthermophilic archaeon was isolated from hydrothermal fluid obtained from Suiyo Seamount of the Izu-Bonin Arc. The isolate, TS1(T), is an irregular coccus, usually 0.5-2 microm in diameter and motile with a polar tuft of flagella. Cells in the exponential phase of growth fused at room temperature in the presence of DNA-intercalating dye to become as large as 5 microm in diameter. Fused cells showed dark spots that moved along in the cytoplasm. Large cells with a similar appearance were also observed upon culture at 87 degrees C, suggesting the occurrence of similar cell fusions during growth. Transmission electron microscopy revealed that cells in the exponential phase possessed a thin and electron-lucent cell envelope that could be lost subsequently during culture. The fragile cell envelope must be related to cell fusion. The cells grew at 57-90 degrees C, pH 5.2-8.7 and at NaCl concentrations of 1.5-4.5 %, with the optima being 87 degrees C, pH 6.5 and 2.5 % NaCl. The isolate was an anaerobic chemo-organotroph that grew on either yeast extract or tryptone as the sole growth substrate. The genomic DNA G+C content was 53.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate was closely related to Thermococcus species. However, no significant DNA-DNA hybridization was observed between genomic DNA of strain TS1(T) and phylogenetically related Thermococcus species. We propose that isolate TS1(T) represents a novel species, Thermococcus coalescens sp. nov., with the name reflecting the cell fusion activity observed in the strain. The type strain is TS1(T) (=JCM 12540T=DSM 16538T).

Analysis of the archaeal sub-seafloor community at Suiyo Seamount on the Izu-Bonin Arc.

A sub-surface archaeal community at the Suiyo Seamount in the Western Pacific Ocean was investigated by 16S rRNA gene sequence and whole-cell in situ hybridization analyses. In this study, we drilled and cased holes at the hydrothermal area of the seamount to minimize contamination of the hydrothermal fluid in the sub-seafloor by penetrating seawater. PCR clone analysis of the hydrothermal fluid samples collected from a cased hole indicated the presence of chemolithoautotrophic primary biomass producers of Archaeoglobales and the Methanococcales-related archaeal HTE1 group, both of which can utilize hydrogen as an electron donor. We discuss the implication of the microbial community on the early history of life and on the search for extraterrestrial life.

Organics in chimneys and water samples from deep-sea hydrothermal systems: implications for sub-vent biosphere.

Searching for life in extreme terrestrial environments can be a model of that for extraterrestrial life. Submarine hydrothermal system is one of promising sites for the frontier of life on the earth. Here seawater and vent chimnies were collected from deep-sea hydrothermal vents at Suiyo Seamount, Izu-bonin arc, Pacific Ocean as a part of Archaean Park Project. Pure seawater sample of 300 degrees C (purity>97%) could be collected. Dissolved and total hydrolyzable amino acids were determined by ion-exchange HPLC, and their enantiomeric ratio was measured by reversed-phase HPLC for the first time. Glycine and serine were two most abundant amino acids, followed by other proteinous amino acids such as alanine, glutamic acid and aspartic acid. Non-proteinous amino acids were detected as minor constituents. Most of the amino acids detected were of the L-form. Thus amino acids of abiotic origin were quite minor, and most of the amino acids detected were formed biologically. These results, together with analytical results of the vent chimney samples, suggest that there is active microbial activities near the hydrothermal systems.

Pyrolysis experiment of simulated exogenous complex organics synthesized from the gas mixtures of CO, NH3, and H2O by 3 MeV proton irradiation.

High molecular weight organic matter synthesized from mixtures of carbon monoxide, ammonia and water gases similar to those found in the interstellar medium were irradiated with a 3 MeV proton beam and analyzed by Curie point pyrolysis with detection by gas chromatograph and mass spectrometer (Pyr-GC-MS). A wide variety of organic compounds, not only a number of amide compounds, but also heterocyclic and polycyclic aromatic hydrocarbons (PAHs), were detected among the products of the pyrolysis. The present data shows that primary and primitive organic matter serving as precursors to bioorganic compounds such as amino acids, nucleic acid bases and sugar might have been formed in a gaseous mixture of similar composition to that of the interstellar dust environment.

Thermanaeromonas toyohensis gen. nov., sp. nov., a novel thermophilic anaerobe isolated from a subterranean vein in the Toyoha Mines.

A novel thermophilic, strictly anaerobic, thiosulfate-reducing bacterium, designated strain ToBE(T), was isolated from a geothermal aquifer at a depth of 550 m in the Toyoha Mines (Hokkaido, Japan). The cells of this bacterium were rod-shaped (0.6 x 2.6 microm), non-motile and sporulating. Strain ToBE(T) was able to grow on formate, lactate, pyruvate or various sugars in the presence of thiosulfate as an electron acceptor. The strain could grow at 55-73 degrees C and pH 5.5-8.5. The optimum temperature and pH for the growth were 70 degrees C and pH 6.5. The G+C content of the DNA was 49.6 mol %. The major quinone and cellular fatty acids were respectively menaquinone-7 and iso-C15:0 and iso-C17:0. Analysis of the 16S rDNA revealed that the isolate was a member of the gram-positive bacteria and was related to the genus Thermoanaerobacter. However, the phylogenetic tree showed that the strain was distant from any other known bacteria, with sequence similarities of less than 90%. On the basis of phenotypic features and phylogenetic analysis, the name Thermanaeromonas toyohensis gen. nov., sp. nov., is proposed for the isolate, with strain ToBE(T) (= DSM 14490T = JCM 11376T) as the type strain.

Distribution and diversity of thermophilic sulfate-reducing bacteria within a Cu-Pb-Zn mine (Toyoha, Japan).

The distribution and diversity of thermophilic sulfate-reducing bacteria at the Cu-Pb-Zn Toyoha underground mine, Japan, were investigated using denaturing gradient gel electrophoresis analysis based on the 16S rRNA gene, and sequence analysis of the dissimilatory sulfite reductase gene. Hydrothermal waters from different boreholes penetrating the Cu-Pb-Zn sulfide veins were collected and concentrated with a sterile filter (pore size: 0.2 mum) at sites A (64 degrees C), B (71 degrees C), and C (48 degrees C). Microbial mats developed at sites A (53 degrees C), B (66 degrees C), and D (73 degrees C) were harvested. The denaturing gel electrophoresis analysis showed 17 bacterial and three archaeal bands including two of spore-forming, Gram-positive sulfate-reducing bacteria, Desulfotomaculum-like 16S rDNA sequences from site B. The phylogenetic analysis of 16 clone families of dissimilatory sulfite reductase genes indicated that they are Desulfotomaculum-, Thermodesulforhabdus-like sequences, and unresolved sequences. We obtained evidence of the diversity and distribution of microbes related to thermophilic sulfate-reducing bacteria within effluent-hydrothermal groundwater and microbial mats in the thermophilic subsurface environment of the Toyoha Mine.