Methylomarinovum tepidoasis sp. nov., a moderately thermophilic methanotroph of the family Methylothermaceae isolated from a deep-sea hydrothermal field.

A novel aerobic methanotrophic bacterium, designated as strain IN45T, was isolated from in situ colonisation systems deployed at the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. IN45T was a moderately thermophilic obligate methanotroph that grew only on methane or methanol at temperatures between 25 and 56 °C (optimum 45-50 °C). It was an oval-shaped, Gram-reaction-negative, motile bacterium with a single polar flagellum and an intracytoplasmic membrane system. It required 1.5-4.0 % (w/v) NaCl (optimum 2-3 %) for growth. The major phospholipid fatty acids were C16 : 1ω7c, C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8. The 16S rRNA gene sequence comparison revealed 99.1 % sequence identity with Methylomarinovum caldicuralii IT-9T, the only species of the genus Methylomarinovum with a validly published name within the family Methylothermaceae. The complete genome sequence of IN45T consisted of a 2.42-Mbp chromosome (DNA G+C content, 64.1 mol%) and a 20.5-kbp plasmid. The genome encodes genes for particulate methane monooxygenase and two types of methanol dehydrogenase (mxaFI and xoxF). Genes involved in the ribulose monophosphate pathway for carbon assimilation are encoded, but the transaldolase gene was not found. The genome indicated that IN45T performs partial denitrification of nitrate to N2O, and its occurrence was indirectly confirmed by N2O production in cultures grown with nitrate. Genomic relatedness indices between the complete genome sequences of IN45T and M. caldicuralii IT-9T, such as digital DNA-DNA hybridisation (51.2 %), average nucleotide identity (92.94 %) and average amino acid identity (93.21 %), indicated that these two methanotrophs should be separated at the species level. On the basis of these results, strain IN45T represents a novel species, for which we propose the name Methylomarinovum tepidoasis sp. nov. with IN45T (=JCM 35101T =DSM 113422T) as the type strain.

Multispecies Populations of Methanotrophic Methyloprofundus and Cultivation of a Likely Dominant Species from the Iheya North Deep-Sea Hydrothermal Field.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab in diffuse-flow areas. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in a biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription data set of INp10 from the biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits that INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help us better understand the ecological strategies of this possibly widespread marine-specific methanotrophic clade. IMPORTANCE The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts, such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab, Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, and yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated that it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus populations suggests that the hydrothermal field is a promising locale at which to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus spp.

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems.

Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the amount of methane (CH4) released into the environment. AMO acts as a sink of CH4, converting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)climate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO events, especially at times when the role of methane in the carbon cycle was more pronounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the BHP compositions were examined for species of the recently described novel Type I methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopentol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel structures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC-BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subsequently, sediments and authigenic carbonates from methane-influenced marine environments were analysed. Most samples also did not contain significant amounts of aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic methanotophic bacteria. However, the BHP composition of the marine samples do point toward the novel MC-BHPs components being potential new biomarkers for AMO.

Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine Ecosystems.

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.

Draft Genome Sequence of the Moderately Halophilic Methanotroph Methylohalobius crimeensis Strain 10Ki.

Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium.

Psychrobium conchae gen. nov., sp. nov., a psychrophilic marine bacterium isolated from the Iheya North hydrothermal field.

A novel psychrophilic, marine, bacterial strain designated BJ-1(T) was isolated from the Iheya North hydrothermal field in the Okinawa Trough off Japan. Cells were Gram-negative, rod-shaped, non-spore-forming, aerobic chemo-organotrophs and motile by means of a single polar flagellum. Growth occurred at temperatures below 16 °C, with the optimum between 9 and 12 °C. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the closest relatives of strain BJ-1(T) were Shewanella denitrificans OS-217(T) (93.5% similarity), Shewanella profunda DSM 15900(T) (92.9%), Shewanella gaetbuli TF-27(T) (92.9%), Paraferrimonas sedimenticola Mok-106(T) (92.1%) and Ferrimonas kyonanensis Asr22-7(T) (91.7%). The major respiratory quinone was Q-8. The predominant fatty acids were C(16:1)ω7c and C(16:0). The G+C content of the novel strain was 40.5 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, it is proposed that strain BJ-1(T) represents a novel species in a new genus, for which the name Psychrobium conchae gen. nov., sp. nov. is proposed. The type strain of Psychrobium conchae is BJ-1(T) ( =JCM 30103(T) =DSM 28701(T)).

Methylomarinovum caldicuralii gen. nov., sp. nov., a moderately thermophilic methanotroph isolated from a shallow submarine hydrothermal system, and proposal of the family Methylothermaceae fam. nov.

A novel methane-oxidizing bacterium, strain IT-9(T), was isolated from a shallow submarine hydrothermal system occurring in a coral reef in Japan. Strain IT-9(T) was a Gram-negative, aerobic, motile, coccoid or oval-shaped bacterium with the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain IT-9(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 30-55 °C (optimum 45-50 °C). The strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. NaCl was required for growth within a concentration range of 1-5 % (optimum 3 %). The hao gene encoding hydroxylamine oxidoreductase (HAO) involved in nitrification was detected by a PCR experiment. The major phospholipid fatty acids were C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8. The DNA G+C content was 66.0 mol%. The 16S rRNA gene sequence of strain IT-9(T) was only moderately related to the sequences of members of the closest genera Methylohalobius (94.1 % similarity) and Methylothermus (91.7-91.9 % similarity); however, those sequences formed a deeply branching monophyletic group within the order Methylococcales. Phylogenies based on 16S rRNA gene sequences, deduced partial PmoA sequences and deduced partial Hao sequences and physiological and chemotaxonomic characteristics revealed that strain IT-9(T) represents a novel species of a new genus, for which the name Methylomarinovum caldicuralii gen. nov., sp. nov. is proposed. The type strain of Methylomarinovum caldicuralii is IT-9(T) ( = JCM 13666(T) = DSM 19749(T)). In addition, we propose a new family, Methylothermaceae fam. nov., in the order Methylococcales, to accommodate the genera Methylothermus, Methylohalobius and Methylomarinovum. The genera Methylothermus and Methylohalobius have been recognized as being distinct from other genera in the methane-oxidizing order Methylococcales in the class Gammaproteobacteria. These genera form a distinctive monophyletic lineage within the order on the basis of 16S rRNA gene sequence phylogeny. This seems consistent with their distinctive physiological traits; the genus Methylothermus includes the most thermophilic species, and the genus Methylohalobius includes the most halophilic species, within the order. Although these two genera include only three species at the time of writing, similar sequences of 16S rRNA genes and pmoA genes encoding pMMO have been detected in a geothermal area or deep-sea hydrothermal vent fields by studies using culture-independent techniques. This suggests that unknown methanotrophs of this lineage inhabit various extreme environments.

Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clam Calyptogena okutanii with thioautotrophic symbiotic bacteria.

Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO2 to synthesize organic compounds. Carbonic anhydrase (CA) from the host catalyzes the reaction CO2 + H2O ↔ HCO3(-) + H(+), and is assumed to facilitate inorganic carbon (Ci) uptake and transport to the symbiont. However, the localization of CA in gill tissue remains unknown. We therefore analyzed mRNA sequences, proteins and CA activity in Calyptogena okutanii using expression sequence tag, SDS-PAGE and LC-MS/MS. We found that acetazolamide-sensitive soluble CA was abundantly expressed in the gill tissue of C. okutanii, and the enzyme was purified by affinity chromatography. Mouse monoclonal antibodies against the CA of C. okutanii were used in western blot analysis and immunofluorescence staining of the gill tissues of C. okutanii, which showed that CA was exclusively localized in the symbiont-harboring cells (bacteriocytes) in gill epithelial cells. Western blot analysis and measurement of activity showed that CA was abundantly (26-72% of total soluble protein) detected in the gill tissues of not only Calyptogena clams but also deep-sea Bathymodiolus mussels that harbor thioautotrophic or methanotrophic symbiotic bacteria, but was not detected in a non-symbiotic mussel, Mytilus sp. The present study showed that CA is abundant in the gill tissues of deep-sea symbiotic bivalves and specifically localizes in the cytoplasm of bacteriocytes of C. okutanii. This indicates that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.

Molecular biological and isotopic biogeochemical prognoses of the nitrification-driven dynamic microbial nitrogen cycle in hadopelagic sediments.

There has been much progress in understanding the nitrogen cycle in oceanic waters including the recent identification of ammonia-oxidizing archaea and anaerobic ammonia oxidizing (anammox) bacteria, and in the comprehensive estimation in abundance and activity of these microbial populations. However, compared with the nitrogen cycle in oceanic waters, there are fewer studies concerning the oceanic benthic nitrogen cycle. To further elucidate the dynamic nitrogen cycle in deep-sea sediments, a sediment core obtained from the Ogasawara Trench at a water depth of 9760 m was analysed in this study. The profiles obtained for the pore-water chemistry, and nitrogen and oxygen stable isotopic compositions of pore-water nitrate in the hadopelagic sediments could not be explained by the depth segregation of nitrifiers and nitrate reducers, suggesting the co-occurrence of nitrification and nitrate reduction in the shallowest nitrate reduction zone. The abundance of SSU rRNA and functional genes related to nitrification and denitrification are consistent with the co-occurrence of nitrification and nitrate reduction observed in the geochemical analyses. This study presents the first example of cooperation between aerobic and anaerobic nitrogen metabolism in the deep-sea sedimentary environments.

Isolation and characterization of a thermophilic, obligately anaerobic and heterotrophic marine Chloroflexi bacterium from a Chloroflexi-dominated microbial community associated with a Japanese shallow hydrothermal system, and proposal for Thermomarinilinea lacunofontalis gen. nov., sp. nov.

A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7(T), was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7(T) was isolated from an enrichment culture at a high dilution rate. Strain SW7(T) was an obligately anaerobic heterotroph that grew with fermentation and had non-motile thin rods 3.5-16.5 µm in length and 0.2 µm in width constituting multicellular filaments. Growth was observed between 37-65°C (optimum 60°C), pH 5.5-7.3 (optimum pH 6.0), and 0.5-3.5% (w/v) NaCl concentration (optimum 1.0%). Based on the physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7(T) (=JCM15506(T)=KCTC5908(T)).

Methylomarinum vadi gen. nov., sp. nov., a methanotroph isolated from two distinct marine environments.

Two aerobic methane-oxidizing bacterial strains were isolated from distinct marine environments in Japan. Strains IT-4(T) and T2-1 were Gram-stain-negative, aerobic, motile, plump short rods or oval-shaped bacteria with a single polar flagellum and type I intracytoplasmic membranes. They were obligate methanotrophs that grew only on methane or methanol. Each strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. The strains grew best at 37 °C, and did not grow at 45 °C. NaCl was required for growth within a concentration range of 1-8 % (w/v). The major phospholipid fatty acids were C16 : 0, C16 : 1ω7c, and C16 : 1ω5t. The major isoprenoid quinone was MQ-8. The DNA G+C content was 50.9-51.7 mol%. The 16S rRNA gene sequences of the strains showed 99.4 % similarity to each other, and DNA-DNA hybridization analysis indicated that the strains were representatives of the same species. The 16S rRNA gene sequences were highly similar to some marine environmental sequences (94.0-97.7 % similarity), but did not show similarities more than 94 % with sequences of members of other related genera, such as Methylomicrobium, Methylobacter, Methylomonas and Methylosarcina. Phylogenies based on 16S rRNA gene sequences and deduced partial PmoA sequences, and the physiological and chemotaxonomic characteristics revealed that strains IT-4(T) and T2-1 represent a novel species of a new genus in the family Methylococcaceae, for which the name Methylomarinum vadi gen. nov., sp. nov. is proposed. The type strain is IT-4(T) ( = JCM 13665(T) = DSM 18976(T)).

Cell-specific thioautotrophic productivity of epsilon-proteobacterial epibionts associated with Shinkaia crosnieri.

In this study, we report experimental evidence of the thioautotrophic activity of the epibiotic microbial community associated with the setae of Shinkaia crosnieri, a galatheid crab that is endemic to deep-sea hydrothermal systems in the Okinawa Trough in Japan. Microbial consumption of reduced sulfur compounds under in situ hydrostatic and atmospheric pressure provided evidence of sulfur-oxidizing activity by the epibiotic microbial community; the rate of sulfur oxidation was similar under in situ and decompressed conditions. Results of the microbial consumption of reduced sulfur compounds and tracer experiments using (13)C-labeled bicarbonate in the presence and absence of thiosulfate (used as a thioautotrophic substrate) convincingly demonstrated that the epibiotic microbial community on S. crosnieri drove primary production via an energy metabolism that was coupled with the oxidation of reductive sulfur compounds. A combination of tracer experiments, fluorescence in situ hybridization (FISH) and nano-scale secondary ion mass spectrometry (Nano-SIMS) indicated that the filamentous cells of the genus Sulfurovum belonging to the class Epsilonproteobacteria were thioautotrophs in the epibiotic community of S. crosnieri. In conclusion, our results strongly suggest that thioautotrophic production by Sulfurovum members present as the epibiotic microbial community play a predominant role in a probable nutritional ectosymbiosis with S. crosnieri.

Methylothermus subterraneus sp. nov., a moderately thermophilic methanotroph isolated from a terrestrial subsurface hot aquifer.

A novel methane-oxidizing bacterium, strain HTM55(T), was isolated from subsurface hot aquifer water from a Japanese gold mine. Strain HTM55(T) was a Gram-negative, aerobic, motile, coccoid bacterium with a single polar flagellum and the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain HTM55(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 37-65 °C (optimum 55-60 °C). The isolate grew at pH 5.2-7.5 (optimum 5.8-6.3) and with 0-1 % NaCl (optimum 0-0.3 %). The ribulose monophosphate pathway was operative for carbon assimilation. The DNA G+C content was 54.4 mol% and the major fatty acids were C(16 : 0) (52.0 %) and C(18 : 1)ω7c (34.8 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HTM55(T) was closely related to Methylothermus thermalis MYHT(T) (99.2 % 16S rRNA gene sequence similarity), which is within the class Gammaproteobacteria. However, DNA-DNA relatedness between strain HTM55(T) and Methylothermus thermalis MYHT(T) was ≤ 39 %. On the basis of distinct phylogenetic, chemotaxonomic and physiological characteristics, strain HTM55(T) represents a novel species of the genus Methylothermus, for which the name Methylothermus subterraneus sp. nov. is proposed. The type strain is HTM55(T) ( = JCM 13664(T) = DSM 19750(T)).

Kosmotoga arenicorallina sp. nov. a thermophilic and obligately anaerobic heterotroph isolated from a shallow hydrothermal system occurring within a coral reef, southern part of the Yaeyama Archipelago, Japan, reclassification of Thermococcoides shengliensis as Kosmotoga shengliensis comb. nov., and emended description of the genus Kosmotoga.

A novel thermophilic and sulfur-reducing bacterium, strain S304(T), was isolated from the Taketomi submarine hot spring shallow hydrothermal field located at southern part of the Yaeyama Archipelago, Japan. The cells were non-motile short thick rods or oval cocci 1.1-2.7 µm in length and 1.1-1.9 µm in width. Strain S304(T) was an obligately anaerobic heterotroph and sulfur reduction stimulates growth. Growth was observed between 50-65°C (optimum 60°C), pH 6.2-8.0 (optimum pH 7.1), 1.0-6.0% NaCl concentration (optimum 3.0%). The fatty acid composition was C(16:0) (71.4%), C(18:0) (20.9%) and C(18:1) (7.7%). The G + C content of genomic DNA was 40.8 mol%. The 16S rRNA gene sequence analysis indicated that strain S304(T) belonged to the genus Kosmotoga. Based on physiological and phylogenetic features of a new isolate, we propose new species in the genus Kosmotoga: the type strain of Kosmotoga arenicorallina sp. nov is S304(T) (=JCM 15790(T) = DSM22549(T)). Thermococcoides shengliensis 2SM-2(T) is phylogenetically associated with Kosmotoga olearia 14.5.1(T). Based on the phylogenetic relationship between Thermococcoides shengliensis 2SM-2(T) and Kosmotoga olearia 14.5.1(T), we propose the reclassification of Thermococcoides shengliensis as Kosmotoga shengliensis comb. nov. (type strain 2SM-2(T)). In addition, an emended description of the genus Kosmotoga is proposed.

Archaeal diversity and distribution along thermal and geochemical gradients in hydrothermal sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa trough.

A variety of archaeal lineages have been identified using culture-independent molecular phylogenetic surveys of microbial habitats occurring in deep-sea hydrothermal environments such as chimney structures, sediments, vent emissions, and chemosynthetic macrofauna. With the exception of a few taxa, most of these archaea have not yet been cultivated, and their physiological and metabolic traits remain unclear. In this study, phylogenetic diversity and distribution profiles of the archaeal genes encoding small subunit (SSU) rRNA, methyl coenzyme A (CoA) reductase subunit A, and the ammonia monooxygenase large subunit were characterized in hydrothermally influenced sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough. Sediment cores were collected at distances of 0.5, 2, or 5 m from a vent emission (90 degrees C). A moderate temperature gradient extends both horizontally and vertically (5 to 69 degrees C), indicating the existence of moderate mixing between the hydrothermal fluid and the ambient sediment pore water. The mixing of reductive hot hydrothermal fluid and cold ambient sediment pore water establishes a wide spectrum of physical and chemical conditions in the microbial habitats that were investigated. Under these different physico-chemical conditions, variability in archaeal phylotype composition was observed. The relationship between the physical and chemical parameters and the archaeal phylotype composition provides important insight into the ecophysiological requirements of uncultivated archaeal lineages in deep-sea hydrothermal vent environments, giving clues for approximating culture conditions to be used in future culturing efforts.

Isolation and physiological characterization of two novel, piezophilic, thermophilic chemolithoautotrophs from a deep-sea hydrothermal vent chimney.

Two novel, thermophilic piezophiles, capable of chemolithoautotrophic growth, are successfully cultivated and isolated from a black smoker chimney at the TAG field (Mid Atlantic Ridge: MAR) by using a piezophilic cultivation technique. Both strains (strains 106 and 108) represent dominant cultivated populations of the microbial communities in the chimney surface habitat. Strain 106 represents typically thin, long spiral cells under the piezophilic growth condition but short bent cells under the non-piezophilic condition. It is a strictly chemolithoautotrophic gammaproteobacterium using reduced sulfur compounds as the electron donors, and nitrate and O(2) as the electron acceptors. Based on the 16S rRNA gene sequence, strain 106 would represent a novel genus of the previously uncultivated group (Symbiont Group I; a potentially novel family) within the Gammaproteobacteria, and 'Thioprofundum lithotrophica' gen. nov., sp. nov. is proposed. Strain 108 is a short, oval rod at any of the growth pressures. It is a facultative chemoautotroph, capable of both chemolithoautotrophic growth with H(2) and S oxidations and organotrophic growth with complex organics or organic acids using nitrate and O(2) as the electron acceptors. The chemolithoautotrophic growth is strictly piezophilic and under the organotrophic growth condition, it grows at conventional pressures (0.1 MPa). Strain 108 is phylogenetically distinctive from any of the previously described genera of the family Rhodobacteraceae within the Alphaproteobacteria, and 'Piezobacter thermophilus' gen. nov., sp. nov. is proposed. The piezophilic cultivation technique can be a powerful tool to isolate and characterize the previously uncultivated phylotypes in the deep-sea hydrothermal vent environments.

Exilispira thermophila gen. nov., sp. nov., an anaerobic, thermophilic spirochaete isolated from a deep-sea hydrothermal vent chimney.

A novel thermophilic, anaerobic bacterium, strain RASEN(T), was isolated from a deep-sea hydrothermal vent chimney sample collected in the Iheya North field, Okinawa Trough, Japan, at a water depth of 982 m. The cells were motile, Gram-negative and helical with hooked ends, 0.23-0.28x15-27 microm in size with an approximate wavelength of 1.1-1.5 microm. Growth of the strain was observed at 37-60 degrees C (optimum 50 degrees C), in 2.5-3.5 % (w/v) NaCl (optimum 2.5-3 % NaCl) and at pH 6.0-7.5 (optimum pH 7.0). The strain grew on yeast extract only of the substrates examined in this study. The G+C content of the genomic DNA was 27.1 mol%. Major fatty acids for the strain were C(16 : 0), C(18 : 1)(Delta9) trans, C(18 : 0) and C(18 : 1)(Delta9) cis. Based on comparative 16S rRNA gene sequence analysis, strain RASEN(T) formed a deeply branching lineage within the phylum Spirochaetes and had only low levels of sequence similarity with other species of the phylum (range of similarity 72.1-80.6 %). Hence, we propose the name Exilispira thermophila gen. nov., sp. nov. The type strain of Exilispira thermophila is strain RASEN(T) (=JCM 14728(T) =NBRC 103205(T) =KCTC 5595(T)).

Cell proliferation at 122 degrees C and isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation.

We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116 degrees C at 0.4 MPa to 122 degrees C at 20 MPa, providing the potential for growth even at 122 degrees C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO(2). Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (-34 per thousand to -27 per thousand) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (< -12 per thousand), and the kinetic isotope effect at 122 degrees C and 40 MPa was -9.4 per thousand, which is one of the smallest effects ever reported. This observation will shed light on the sources and production mechanisms of deep-sea methane.

Culture-dependent and -independent characterization of microbial communities associated with a shallow submarine hydrothermal system occurring within a coral reef off Taketomi Island, Japan.

Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, approximately 23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52 degrees C) contained 175 microM H2S and gas bubbles mainly composed of CH4 (69%) and N2 (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37 degrees C, and another novel type I methanotroph exhibited optimum growth at 45 degrees C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.

Microbial community in a sediment-hosted CO2 lake of the southern Okinawa Trough hydrothermal system.

Increasing levels of CO2 in the atmosphere are expected to cause climatic change with negative effects on the earth's ecosystems and human society. Consequently, a variety of CO2 disposal options are discussed, including injection into the deep ocean. Because the dissolution of CO2 in seawater will decrease ambient pH considerably, negative consequences for deep-water ecosystems have been predicted. Hence, ecosystems associated with natural CO2 reservoirs in the deep sea, and the dynamics of gaseous, liquid, and solid CO2 in such environments, are of great interest to science and society. We report here a biogeochemical and microbiological characterization of a microbial community inhabiting deep-sea sediments overlying a natural CO2 lake at the Yonaguni Knoll IV hydrothermal field, southern Okinawa Trough. We found high abundances (>10(9) cm(-3)) of microbial cells in sediment pavements above the CO2 lake, decreasing to strikingly low cell numbers (10(7) cm(-3)) at the liquid CO2/CO2-hydrate interface. The key groups in these sediments were as follows: (i) the anaerobic methanotrophic archaea ANME-2c and the Eel-2 group of Deltaproteobacteria and (ii) sulfur-metabolizing chemolithotrophs within the Gamma- and Epsilonproteobacteria. The detection of functional genes related to one-carbon assimilation and the presence of highly 13C-depleted archaeal and bacterial lipid biomarkers suggest that microorganisms assimilating CO2 and/or CH4 dominate the liquid CO2 and CO2-hydrate-bearing sediments. Clearly, the Yonaguni Knoll is an exceptional natural laboratory for the study of consequences of CO2 disposal as well as of natural CO2 reservoirs as potential microbial habitats on early Earth and other celestial bodies.