Group-Specific 16S rRNA-Targeted Oligonucleotide Probes To Identify Thermophilic Bacteria in Marine Hydrothermal Vents

Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems. We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the genus Thermus, the genera Thermotoga and Thermosipho, and the Aquificales order. The temperature of dissociation of each probe was determined. Probe specificities to the target groups were demonstrated by whole-cell and dot blot hybridization against a collection of target and nontarget rRNAs. Whole-cell hybridizations with the specific probes were performed on cells extracted from hydrothermal vent chimneys. One of the samples contained cells that hybridized to the probe specific to genera Thermotoga and Thermosipho. No positive signals could be detected in the samples tested with the probes whose specificities encompassed either the genus Thermus or the thermophilic members of the genus Bacillus. However, when simultaneous hybridizations with the probe specific to the order Aquificales and a probe specific to the domain Bacteria (R. I. Amann, B. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl, Appl. Environ. Microbiol. 56:1919-1925, 1990) were performed on cells extracted from the top and exterior subsamples of chimneys, positive signals were obtained from morphologically diverse bacteria representing about 40% of the bacterial population. Since specificity studies also revealed that the bacterial probe did not hybridize with the members of the order Aquificales, the detected cells may therefore correspond to a new type of bacteria. One of the observed morphotypes was similar to that of a strictly anaerobic autotrophic sulfur-reducing strain that we isolated from the chimney samples. This work demonstrates that application of whole-cell hybridization with probes specific for different phylogenetic levels is a useful tool for detailed studies of hydrothermal vent microbial ecology.     

Comparative metagenomics of microbial communities inhabiting deep-sea hydrothermal vent chimneys with contrasting chemistries

Deep-sea hydrothermal vent chimneys harbor a high diversity of largely unknown microorganisms. Although the phylogenetic diversity of these microorganisms has been described previously, the adaptation and metabolic potential of the microbial communities is only beginning to be revealed. A pyrosequencing approach was used to directly obtain sequences from a fosmid library constructed from a black smoker chimney 4143-1 in the Mothra hydrothermal vent field at the Juan de Fuca Ridge. A total of 308 034 reads with an average sequence length of 227 bp were generated. Comparative genomic analyses of metagenomes from a variety of environments by two-way clustering of samples and functional gene categories demonstrated that the 4143-1 metagenome clustered most closely with that from a carbonate chimney from Lost City. Both are highly enriched in genes for mismatch repair and homologous recombination, suggesting that the microbial communities have evolved extensive DNA repair systems to cope with the extreme conditions that have potential deleterious effects on the genomes. As previously reported for the Lost City microbiome, the metagenome of chimney 4143-1 exhibited a high proportion of transposases, implying that horizontal gene transfer may be a common occurrence in the deep-sea vent chimney biosphere. In addition, genes for chemotaxis and flagellar assembly were highly enriched in the chimney metagenomes, reflecting the adaptation of the organisms to the highly dynamic conditions present within the chimney walls. Reconstruction of the metabolic pathways revealed that the microbial community in the wall of chimney 4143-1 was mainly fueled by sulfur oxidation, putatively coupled to nitrate reduction to perform inorganic carbon fixation through the Calvin–Benson–Bassham cycle. On the basis of the genomic organization of the key genes of the carbon fixation and sulfur oxidation pathways contained in the large genomic fragments, both obligate and facultative autotrophs appear to be present and contribute to biomass production.     

Assessing the influence of physical,geochemical and biological factors on anaerobic microbial primary productivity within hydrothermal vent chimneys

Chemosynthetic primary production supports hydrothermal vent ecosystems, but the extent of that productivity and its governing factors have not been well constrained. To better understand anaerobic primary production within massive vent deposits, we conducted a series of incubations at 4, 25, 50 and 90 °C using aggregates recovered from hydrothermal vent structures. We documented in situ geochemistry, measured autochthonous organic carbon stable isotope ratios and assessed microbial community composition and functional gene abundances in three hydrothermal vent chimney structures from Middle Valley on the Juan de Fuca Ridge. Carbon fixation rates were greatest at lower temperatures and were comparable among chimneys. Stable isotope ratios of autochthonous organic carbon were consistent with the Calvin–Benson–Bassham cycle being the predominant mode of carbon fixation for all three chimneys. Chimneys exhibited marked differences in vent fluid geochemistry and microbial community composition, with structures being differentially dominated by gamma (γ) or epsilon (ε) proteobacteria. Similarly, qPCR analyses of functional genes representing different carbon fixation pathways showed striking differences in gene abundance among chimney structures. Carbon fixation rates showed no obvious correlation with observed in situ vent fluid geochemistry, community composition or functional gene abundance. Together, these data reveal that (i) net anaerobic carbon fixation rates among these chimneys are elevated at lower temperatures, (ii) clear differences in community composition and gene abundance exist among chimney structures, and (iii) tremendous spatial heterogeneity within these environments likely confounds efforts to relate the observed rates to in situ microbial and geochemical factors. We also posit that microbes typically thought to be mesophiles are likely active and growing at cooler temperatures, and that their activity at these temperatures comprises the majority of endolithic anaerobic primary production in hydrothermal vent chimneys.     

Continuous enrichment cultures: insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys

The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulfatator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO(2) and H(2) produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments.     

Microbial Diversity in Inactive Chimney Structures from Deep-Sea Hydrothermal Systems

Massive chimney structures, which are characteristic of many hydrothermally active zones, harbor diverse microbial communities containing both thermophilic and hyperthermophilic microbes. However, vent chimneys ultimately become hydrothermally inactive, and the changes that occur in the microbial communities upon becoming inactive have not been documented. We thus collected inactive chimneys from two geologically and geographically distinct hydrothermal fields, Iheya North in the western Pacific Ocean and the Kairei field in the Indian Ocean. The chimneys displayed easily distinguishable strata, which were analyzed with regard to both mineralogical and microbiological properties. X-ray diffraction pattern and energy-dispersive spectroscopic analyses revealed that the main mineral components of the chimney substructures from Iheya North and the Kairei field were barite (BaSO₄) and chalcopyrite (CuFeS₂), respectively. Microbial cell densities in the substructures determined by DAPI counting ranged from 1.7 × 10⁷ cells g^–1 to 3.0 × 10⁸ cells g^–1 . The proportions of archaeal rDNA in the whole microbial rDNA assemblages in all substructures were, at most, a few percent as determined by quantitative fluorogenic PCR. The microbial rDNA clone analysis and whole-cell fluorescence in situ hybridization revealed a community that was decidedly different from any communities previously reported in active chimneys. Curiously, both samples revealed the abundant presence of a group of Bacteria related to a magnetosome-bearing bacterium, Magnetobacterium bavaricum of the Nitrospirae division. These results suggest that inactive chimneys provide a distinct microbial habitat.     

Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney

A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998. Finn was venting 302 degrees C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit. Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney. The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >10(8) cells/g of sulfide 2 to 10 cm within the chimney wall to <10(5) cells/g in interior zones. Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure. Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior. Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene. The majority of sequences from the exterior of the chimney were related to marine group I of the Crenarchaeota and uncultured Euryarchaeota from benthic marine environments. In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens, Thermococcales, and Archaeoglobales, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites. These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life.     

Effect of variation of environmental conditions on the microbial communities of deep-sea vent chimneys, cultured in a bioreactor

Both cultivation and molecular techniques were used to investigate the microbial diversity and dynamic of a deep-sea vent chimney. The enrichment cultures performed in a gas-lift bioreactor were inoculated with a black smoker chimney sample collected on TAG site on the mid-Atlantic ridge. To mimic as close as possible environmental conditions, the cultures were performed in oligotrophic medium with nitrogen, hydrogen and carbon dioxide (N₂/H₂/CO₂) gas sweeping. Also, the temperature was first settled at a temperature of 85°C and colloidal sulphur was added. Then, the temperature was lowered to 60°C and sulphur was omitted. Archaeal and bacterial diversity was studied in both culture and natural samples. Through 16S rRNA gene sequences analysis of the enrichment cultures microorganisms affiliated to Archeoglobales, Thermococcales were detected in both conditions while, Deferribacterales and Thermales were detected only at 65°C in the absence of sulphur. Single-stranded conformational polymorphism and quantitative PCR permit to study the microbial community dynamic during the two enrichment cultures. The effect of environmental changes (modification of culture conditions), i.e. temperature, medium composition, electron donors and acceptors availability were shown to affect the microbial community in culture, as this would happen in their environment. The effect of environmental changes, i.e. temperature and medium composition was shown to affect the microbial community in culture, as this could happen in their environment. The modification of culture conditions, such as temperature, organic matter concentration, electron donors and acceptors availability allowed to enrich different population of prokaryotes inhabiting hydrothermal chimneys.     

Microbial diversity of Loki's Castle black smokers at the Arctic Mid‐Ocean Ridge

Hydrothermal vent systems harbor rich microbial communities ranging from aerobic mesophiles to anaerobic hyperthermophiles. Among these, members of the archaeal domain are prevalent in microbial communities in the most extreme environments, partly because of their temperature‐resistant and robust membrane lipids. In this study, we use geochemical and molecular microbiological methods to investigate the microbial diversity in black smoker chimneys from the newly discovered Loki's Castle hydrothermal vent field on the Arctic Mid‐Ocean Ridge (AMOR) with vent fluid temperatures of 310–320 °C and pH of 5.5. Archaeal glycerol dialkyl glycerol tetraether lipids (GDGTs) and H‐shaped GDGTs with 0–4 cyclopentane moieties were dominant in all sulfide samples and are most likely derived from both (hyper)thermophilic Euryarchaeota and Crenarchaeota. Crenarchaeol has been detected in low abundances in samples derived from the chimney exterior indicating the presence of Thaumarchaeota at lower ambient temperatures. Aquificales and members of the Epsilonproteobacteria were the dominant bacterial groups detected. Our observations based on the analysis of 16S rRNA genes and biomarker lipid analysis provide insight into microbial communities thriving within the porous sulfide structures of active and inactive deep‐sea hydrothermal vents. Microbial cycling of sulfur, hydrogen, and methane by archaea in the chimney interior and bacteria in the chimney exterior may be the prevailing biogeochemical processes in this system.     

Incidence and Diversity of Microorganisms within the Walls of an Active Deep-Sea Sulfide Chimney

A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998. Finn was venting 302°C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit. Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney. The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >10⁸ cells/g of sulfide 2 to 10 cm within the chimney wall to <10⁵ cells/g in interior zones. Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure. Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior. Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene. The majority of sequences from the exterior of the chimney were related to marine group I of the Crenarchaeota and uncultured Euryarchaeota from benthic marine environments. In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens, Thermococcales, and Archaeoglobales, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites. These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life.     

Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge

The bacterial and archaeal communities of three deep-sea hydrothermal vent systems located on the Mid-Atlantic Ridge (MAR; Rainbow, Logatchev and Broken Spur) were investigated using an integrated culture-dependent and independent approach. Comparative molecular phylogenetic analyses, using the 16S rRNA gene and the deduced amino acid sequences of the alpha and beta subunits of the ATP citrate lyase encoding genes were carried out on natural microbial communities, on an enrichment culture obtained from the Broken Spur chimney, and on novel chemolithoautotrophic bacteria and reference strains originally isolated from several different deep-sea vents. Our data showed that the three MAR hydrothermal vent chimneys investigated in this study host very different microbial assemblages. The microbial community of the Rainbow chimney was dominated by thermophilic, autotrophic, hydrogen-oxidizing, sulfur- and nitrate-reducing Epsilonproteobacteria related to the genus Caminibacter. The detection of sequences related to sulfur-reducing bacteria and archaea (Archaeoglobus) indicated that thermophilic sulfate reduction might also be occurring at this site. The Logatchev bacterial community included several sequences related to mesophilic sulfur-oxidizing bacteria, while the archaeal component of this chimney was dominated by sequences related to the ANME-2 lineage, suggesting that anaerobic oxidation of methane may be occurring at this site. Comparative analyses of the ATP citrate lyase encoding genes from natural microbial communities suggested that Epsilonproteobacteria were the dominant primary producers using the reverse TCA cycle (rTCA) at Rainbow, while Aquificales of the genera Desulfurobacterium and Persephonella were prevalent in the Broken Spur chimney.     

Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano,Hellenic Volcanic Arc

Over the last decades, there has been growing interest about the ecological role of hydrothermal sulfide chimneys, their microbial diversity and associated biotechnological potential. Here, we performed dual-index Illumina sequencing of bacterial and archaeal communities on active and inactive sulfide chimneys collected from the Kolumbo hydrothermal field, situated on a geodynamic convergent setting. A total of 15,701 OTUs (operational taxonomic units) were assigned to 56 bacterial and 3 archaeal phyla, 133 bacterial and 16 archaeal classes. Active chimney communities were dominated by OTUs related to thermophilic members of Epsilonproteobacteria, Aquificae and Deltaproteobacteria. Inactive chimney communities were dominated by an OTU closely related to the archaeon Nitrosopumilus sp., and by members of Gammaproteobacteria, Deltaproteobacteria, Planctomycetes and Bacteroidetes. These lineages are closely related to phylotypes typically involved in iron, sulfur, nitrogen, hydrogen and methane cycling. Overall, the inactive sulfide chimneys presented highly diverse and uniform microbial communities, in contrast to the active chimney communities, which were dominated by chemolithoautotrophic and thermophilic lineages. This study represents one of the most comprehensive investigations of microbial diversity in submarine chimneys and elucidates how the dissipation of hydrothermal activity affects the structure of microbial consortia in these extreme ecological niches.

     

Temporal changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-microm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.     

Temporal Changes in Archaeal Diversity and Chemistry in a Mid-Ocean Ridge Subseafloor Habitat

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-μm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.     

Lipid Biomarkers Reveal Microbial Communities in Hydrothermal Chimney Structures from the 49.6°E Hydrothermal Vent Field at the Southwest Indian Ocean Ridge

Lipid biomarkers were investigated to reveal the microbial life preserved in sulfide and Si-rich chimney from the 49.6°E hydrothermal vent field. In sulfide chimney, iso-/anteiso-fatty acids and H-shaped glycerol dialkyl glycerol tetraethers are the main microbial biomarkers. In Si-rich chimney, monounsaturated fatty acids (C16:1n7, C18:1n7) are the main bacterial biomarkers detected, and crenarchaeol and its isomer are relatively abundant (up to 25% of glycerol dialkyl glycerol tetraethers) archaeol biomarkers. Composition of lipid biomarkers reveals the diversity of microbial communities in different types of chimney structures. Sulfate-reducing bacteria and hyperthermophilic archaea were considered to be the majority microbial life in sulfide chimney, and sulfur-oxidizing bacteria were abundant in Si-rich chimney while archaea in Si-rich chimney and mainly attributed to Thaumarchaeota, which were predominately ammonia oxidizers. Our result suggested that fluid temperature and gaseous components could be the main constraints for the diversity of microbial communities in hydrothermal chimney structures in 49.6°E hydrothermal vent field.     

Spatial distribution of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.     

Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal lineages in high-temperature environments

In order to facilitate the evaluation of archaeal community diversity and distribution in high-temperature environments, 14 16S rRNA oligonucleotide probes were designed. Adequate hybridization and wash conditions of the probes encompassing most known hyperthermophilic Archaea, members of the orders Thermococcales, Desulfurococcales and Sulfolobales, of the families Methanocaldococcaceae, Pyrodictiaceae and Thermoproteaceae, of the genera Archaeoglobus, Methanopyrus and Ignicoccus, and of the as yet uncultured lineages Korarchaeota, Crenarchaeota marine group I, deep-sea hydrothermal vent euryarchaeotic group 2 (DHVE 2), and deep-sea hydrothermal vent euryarchaeotic group 8 (DHVE 8) were determined by dot-blot hybridization from target and non-target reference organisms and environmental clones. The oligonucleotide probes were also used to evaluate the archaeal community composition in nine deep-sea hydrothermal vent samples. All probes, except those targeting members of Sulfolobales, Thermoproteaceae, Pyrodictiaceae and Korarchaeota, gave positive hybridization signals when hybridized against 16S rDNA amplification products obtained from hydrothermal DNA extracts. The results confirmed the widespread occurrence of Thermococcales, Desulfurococcales, Methanocaldococcaceae and Archaeoglobus in deep-sea hydrothermal vents, and extended the known ecological habitats of uncultured lineages. Despite their wide coverage, the probes were unable to resolve the archaeal communities associated with hydrothermally influenced sediments, suggesting that these samples may contain novel lineages. This suite of oligonucleotide probes may represent an efficient tool for rapid qualitative and quantitative characterization of archaeal communities. Their application would help to provide new insights in the future into the composition, distribution and abundance of Archaea in high-temperature environments.     

Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage

Active deep-sea hydrothermal vents are areas of intense mixing and severe thermal and chemical gradients, fostering a biotope rich in novel hyperthermophilic microorganisms and metabolic pathways. The goal of this study was to identify the earliest archaeal colonizers of nascent hydrothermal chimneys, organisms that may be previously uncharacterized as they are quickly replaced by a more stable climax community. During expeditions in 2001 and 2002 to the hydrothermal vents of the East Pacific Rise (EPR) (9 degrees 50'N, 104 degrees 17'W), we removed actively venting chimneys and in their place deployed mineral chambers and sampling units that promoted the growth of new, natural hydrothermal chimneys and allowed their collection within hours of formation. These samples were compared with those collected from established hydrothermal chimneys from EPR and Guaymas Basin vent sites. Using molecular and phylogenetic analysis of the 16S rDNA, we show here that at high temperatures, early colonization of a natural chimney is dominated by members of the archaeal genus Ignicoccus and its symbiont, Nanoarchaeum. We have identified 19 unique sequences closely related to the nanoarchaeal group, and five archaeal sequences that group closely with Ignicoccus. These organisms were found to colonize a natural, high temperature protochimney and vent-like mineral assemblages deployed over high temperature outflows within 92 h. When compared phylogenetically, several of these colonizing organisms form a unique clade independent of those found in mature chimneys and low-temperature mineral chamber samples. As a model ecosystem, the identification of pioneering consortia in deep-sea hydrothermal vents may help advance the understanding of how early microbial life forms gained a foothold in hydrothermal systems on early Earth and potentially on other planetary bodies.     

Spatial Distribution of Marine Crenarchaeota Group I in the Vicinity of Deep-Sea Hydrothermal Systems

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.     

Geomicrobiological exploration and characterization of a novel deep-sea hydrothermal system at the TOTO caldera in the Mariana Volcanic Arc

Novel hydrothermal activities accompanying effluent white smokers and elemental sulfur chimney structures at the north-east lava dome of the TOTO caldera depression in the Mariana Volcanic Arc have been explored and characterized by geochemical and microbiological surveys. White smoker hydrothermal fluids were observed in the potential hydrothermal activity centre of the field and represented the maximal temperature of 170 degrees C and the lowest pH of 1.6. The chimney structures, all consisting of elemental sulfur (sulfur chimney), were also unique to the TOTO caldera hydrothermal field. Microbial community structures in a sulfur chimney and its formation hydrothermal fluid with a high concentration of hydrogen sulfide (15 mM) have been investigated by culture-dependent and -independent analyses. 16S rRNA gene clone analysis and fluorescence in situ hybridization (FISH) analysis revealed that epsilon-Proteobacteria dominated the microbial communities in the sulfur chimney structure and formed a dense microbial mat covering the sulfur chimney surface. Archaeal phylotypes were consistently minor components in the communities and related to the genera Thermococcus, Pyrodictium, Aeropyrum, and the uncultivated archaeal group of 'deep-sea hydrothermal vent euryarchaeotal group'. Cultivation analysis suggested that the chemolithoautotrophs might play a significant ecological role as primary producers utilizing gas and sulfur compounds provided from hydrothermal fluids.