Phylogenetic Diversity of Nitrogenase (nifH) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N₂ gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea. All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Incidence of novel and potentially archaeal nitrogenase genes in the deep Northeast Pacific Ocean

Archaea have been detected throughout the oceanic water column and are quantitatively important members of picoplankton in the deep ocean. Two common groups, group I Crenarchaeota and group II Euryarchaeota, are consistently detected in warm hydrothermal fluid and are assumed to have been drawn into the subseafloor, mixed with hydrothermal fluid and then expelled. However, because they remain resistant to cultivation, very little is known about their physiology. Here we show that cold deep-seawater from the axial valley of Endeavour Segment on the Juan de Fuca Ridge contains not only groups I and II archaea as expected, but also unique potentially archaeal nitrogenase (nifH) genes, which are required for nitrogen fixation. These nifH genes are phylogenetically distinct and have dissimilar G+C content compared with those of hydrothermal vent archaea, suggesting that they belong to non-thermophilic deep-sea archaea. Furthermore, this sample did not contain mcrA genes, which are present in methanogens, the only known archaeal nitrogen fixers. These nifH genes were not detected in upper water column samples, or in a deep-seawater sample 100 km away from the spreading axis of the Juan de Fuca Ridge. We propose that these unique nifH genes may be localized to archaea that circulate through the nitrogen-poor subseafloor at the mid-ocean ridge as part of their life cycle.     

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.     

Strain-level genomic variation in natural populations of Lebetimonas from an erupting deep-sea volcano

Chemolithoautotrophic Epsilonproteobacteria are ubiquitous in sulfidic, oxygen-poor habitats, including hydrothermal vents, marine oxygen minimum zones, marine sediments and sulfidic caves and have a significant role in cycling carbon, hydrogen, nitrogen and sulfur in these environments. The isolation of diverse strains of Epsilonproteobacteria and the sequencing of their genomes have revealed that this group has the metabolic potential to occupy a wide range of niches, particularly at dynamic deep-sea hydrothermal vents. We expand on this body of work by examining the population genomics of six strains of Lebetimonas, a vent-endemic, thermophilic, hydrogen-oxidizing Epsilonproteobacterium, from a single seamount in the Mariana Arc. Using Lebetimonas as a model for anaerobic, moderately thermophilic organisms in the warm, anoxic subseafloor environment, we show that genomic content is highly conserved and that recombination is limited between closely related strains. The Lebetimonas genomes are shaped by mobile genetic elements and gene loss as well as the acquisition of novel functional genes by horizontal gene transfer, which provide the potential for adaptation and microbial speciation in the deep sea. In addition, these Lebetimonas genomes contain two operons of nitrogenase genes with different evolutionary origins. Lebetimonas expressed nifH during growth with nitrogen gas as the sole nitrogen source, thus providing the first evidence of nitrogen fixation in any Epsilonproteobacteria from deep-sea hydrothermal vents. In this study, we provide a comparative overview of the genomic potential within the Nautiliaceae as well as among more distantly related hydrothermal vent Epsilonproteobacteria to broaden our understanding of microbial adaptation and diversity in the deep sea.     

Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.     

Methane- and sulfur-metabolizing microbial communities dominate the Lost City hydrothermal field ecosystem

Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.     

Abundance of reverse tricarboxylic acid cycle genes in free-living microorganisms at deep-sea hydrothermal vents

Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem. However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of Proteobacteria and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate. Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents. PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples. These genes were also expressed in at least one chimney sample. Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of Proteobacteria. These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of Proteobacteria in free-living hydrothermal vent communities. Members of the epsilon subdivision of Proteobacteria are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface. Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.     

Individual hydrothermal vents at Axial Seamount harbor distinct subseafloor microbial communities

The microbial community structure of five geographically distinct hydrothermal vents located within the Axial Seamount caldera, Juan de Fuca Ridge, was examined over 6 years following the 1998 diking eruptive event. Terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene sequence analyses were used to determine the bacterial and archaeal diversity, and the statistical software primer v6 was used to compare vent microbiology, temperature and fluid chemistry. Statistical analysis of vent fluid temperature and composition shows that there are significant differences between vents in any year, but that the fluid composition changes over time such that no vent maintains a chemical composition completely distinct from the others. In contrast, the subseafloor microbial communities associated with individual vents changed from year to year, but each location maintained a distinct community structure (based on TRFLP and 16S rRNA gene sequence analyses) that was significantly different from all other vents included in this study. Epsilonproteobacterial microdiversity is shown to be important in distinguishing vent communities, while archaeal microdiversity is less variable between sites. We propose that persistent venting at diffuse flow vents over time creates the potential to isolate and stabilize diverse microbial community structures between vents.     

A Comprehensive Evaluation of PCR Primers to Amplify the nifH Gene of Nitrogenase

The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples.     

Halomonas and Marinobacter ecotypes from hydrothermal vent, subseafloor and deep-sea environments

Moderately halophilic and euryhaline bacteria are routinely found in cool to warm hydrothermal vent and nearby cold, deep-sea environments. To elucidate the diversity of these microorganisms - with the goal of determining which among them constitute ecotypes specifically associated with hydrothermal vent and subseafloor habitats - PCR primers were designed to detect natural populations of euryhaline Gammaproteobacteria belonging to the cosmopolitan genera Halomonas and Marinobacter. The distribution patterns of 16S rRNA gene sequence data revealed that Halomonas group 2A comprised a subseafloor population at Axial Seamount on the Juan de Fuca Ridge. Complementary biogeographic and physiological data suggested that other Halomonas clades include members that are cold adapted (Halomonas group 2B) or associated with massive sulfide deposits (Halomonas group 2C). Similarly, a monophyletic Marinobacter clade may represent Fe(2+) -oxidizing facultative chemoautotrophs based on the phylogenetic data presented here and previously reported phenotypic characterizations. The biogeographic distributions of Halomonas and Marinobacter isolates and clones reveal that these are cosmopolitan genera, commonly found in the deep sea and in hydrothermal vent settings. As such, they are good candidates for further laboratory investigations into the biogeochemical processes in these environments.     

Diversity and expression of nitrogen fixation genes in bacterial symbionts of marine sponges

Marine sponges contain complex assemblages of bacterial symbionts, the roles of which remain largely unknown. We identified diverse bacterial nifH genes within sponges and found that nifH genes are expressed in sponges. This is the first demonstration of the expression of any protein-coding bacterial gene within a sponge. Two sponges Ircinia strobilina and Mycale laxissima were collected from Key Largo, Florida and had delta(15)N values of c. 0-1 per thousand and 3-4 per thousand respectively. The potential for nitrogen fixation by symbionts was assessed by amplification of nifH genes. Diverse nifH genes affiliated with Proteobacteria and Cyanobacteria were detected, and expression of nifH genes affiliated with those from cyanobacteria was detected. The nifH genes from surrounding seawater were similar to those of Trichodesmium and clearly different from the cyanobacterial nifH genes detected in the two sponges. This study advances understanding of the role of bacterial symbionts in sponges and suggests that provision of fixed nitrogen is a means whereby symbionts benefit sponges in nutrient-limited reef environments. Nitrogen fixation by sponge symbionts is possibly an important source of new nitrogen to the reef environment that heretofore has been neglected and warrants further investigation.     

Microbial Community Stratification Controlled by the Subseafloor Fluid Flow and Geothermal Gradient at the Iheya North Hydrothermal Field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331)

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments.     

Abundance of Reverse Tricarboxylic Acid Cycle Genes in Free-Living Microorganisms at Deep-Sea Hydrothermal Vents

Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem. However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of Proteobacteria and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate. Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents. PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples. These genes were also expressed in at least one chimney sample. Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of Proteobacteria. These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of Proteobacteria in free-living hydrothermal vent communities. Members of the epsilon subdivision of Proteobacteria are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface. Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.     

Methane- and Sulfur-Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem

Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.     

Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge

To evaluate the effects of local fluid geochemistry on microbial communities associated with active hydrothermal vent deposits, we examined the archaeal and bacterial communities of 12 samples collected from two very different vent fields: the basalt-hosted Lucky Strike (37°17'N, 32°16.3'W, depth 1600-1750 m) and the ultramafic-hosted Rainbow (36°13'N, 33°54.1'W, depth 2270-2330 m) vent fields along the Mid-Atlantic Ridge (MAR). Using multiplexed barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA genes, we show statistically significant differences between the archaeal and bacterial communities associated with the different vent fields. Quantitative polymerase chain reaction (qPCR) assays of the functional gene diagnostic for methanogenesis (mcrA), as well as geochemical modelling to predict pore fluid chemistries within the deposits, support the pyrosequencing observations. Collectively, these results show that the less reduced, hydrogen-poor fluids at Lucky Strike limit colonization by strict anaerobes such as methanogens, and allow for hyperthermophilic microaerophiles, like Aeropyrum. In contrast, the hydrogen-rich reducing vent fluids at the ultramafic-influenced Rainbow vent field support the prevalence of methanogens and other hydrogen-oxidizing thermophiles at this site. These results demonstrate that biogeographical patterns of hydrothermal vent microorganisms are shaped in part by large scale geological and geochemical processes.     

Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.     

Anaerobic Respiration on Tellurate and Other Metalloids in Bacteria from Hydrothermal Vent Fields in the Eastern Pacific Ocean

This paper reports the discovery of anaerobic respiration on tellurate by bacteria isolated from deep ocean (1,543 to 1,791 m) hydrothermal vent worms. The first evidence for selenite- and vanadate-respiring bacteria from deep ocean hydrothermal vents is also presented. Enumeration of the anaerobic metal(loid)-resistant microbial community associated with hydrothermal vent animals indicates that a greater proportion of the bacterial community associated with certain vent fauna resists and reduces metal(loid)s anaerobically than aerobically, suggesting that anaerobic metal(loid) respiration might be an important process in bacteria that are symbiotic with vent fauna. Isolates from Axial Volcano and Explorer Ridge were tested for their ability to reduce tellurate, selenite, metavanadate, or orthovanadate in the absence of alternate electron acceptors. In the presence of metal(loid)s, strains showed an ability to grow and produce ATP, whereas in the absence of metal(loid)s, no growth or ATP production was observed. The protonophore carbonyl cyanide m-chlorophenylhydrazone depressed metal(loid) reduction. Anaerobic tellurate respiration will be a significant component in describing biogeochemical cycling of Te at hydrothermal vents.     

Anaerobic respiration on tellurate and other metalloids in bacteria from hydrothermal vent fields in the eastern Pacific Ocean

This paper reports the discovery of anaerobic respiration on tellurate by bacteria isolated from deep ocean (1,543 to 1,791 m) hydrothermal vent worms. The first evidence for selenite- and vanadate-respiring bacteria from deep ocean hydrothermal vents is also presented. Enumeration of the anaerobic metal(loid)-resistant microbial community associated with hydrothermal vent animals indicates that a greater proportion of the bacterial community associated with certain vent fauna resists and reduces metal(loid)s anaerobically than aerobically, suggesting that anaerobic metal(loid) respiration might be an important process in bacteria that are symbiotic with vent fauna. Isolates from Axial Volcano and Explorer Ridge were tested for their ability to reduce tellurate, selenite, metavanadate, or orthovanadate in the absence of alternate electron acceptors. In the presence of metal(loid)s, strains showed an ability to grow and produce ATP, whereas in the absence of metal(loid)s, no growth or ATP production was observed. The protonophore carbonyl cyanide m-chlorophenylhydrazone depressed metal(loid) reduction. Anaerobic tellurate respiration will be a significant component in describing biogeochemical cycling of Te at hydrothermal vents.