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Physiological and gene expression studies of deep-sea bacteria under pressure conditions similar to those experienced in their natural habitat are critical for understanding growth kinetics and metabolic adaptations to in situ conditions. The Campylobacterium (aka Epsilonproteobacterium) Nautilia sp. strain PV-1 was isolated from hydrothermal fluids released from an active deep-sea hydrothermal vent at 9° N on the East Pacific Rise. Strain PV-1 is a piezophilic, moderately thermophilic, chemolithoautotrophic anaerobe that conserves energy by coupling the oxidation of hydrogen to the reduction of nitrate or elemental sulfur. Using a high-pressure-high temperature continuous culture system, we established that strain PV-1 has the shortest generation time of all known piezophilic bacteria and we investigated its protein expression pattern in response to different hydrostatic pressure regimes. Proteogenomic analyses of strain PV-1 grown at 20 and 5 MPa showed that pressure adaptation is not restricted to stress response or homeoviscous adaptation but extends to enzymes involved in central metabolic pathways. Protein synthesis, motility, transport, and energy metabolism are all affected by pressure, although to different extents. In strain PV-1, low-pressure conditions induce the synthesis of phage-related proteins and an overexpression of enzymes involved in carbon fixation.

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Labonte, J. M., Pachiadaki, M., Fergusson, E., McNichol, J., Grosche, A., Gulmann, L. K., Vetriani, C., Sievert, S. M., & Stepanauskas, R. Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system. Frontiers in Microbiology, 10, (2019): 1262, doi:10.3389/fmicb.2019.01262., Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds., This work was supported by the U.S. National Science Foundation’s Dimensions of Biodiversity Program [OCE-1136488 (to RS), OCE-1136727 (to SMS) and OCE-1136451 (to CV)], as well as DEB-1441717 and OCE-1335810 (to RS), and the DOE JGI CSP project 1477.

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Labonte, J. M., Pachiadaki, M., Fergusson, E., McNichol, J., Grosche, A., Gulmann, L. K., Vetriani, C., Sievert, S. M., & Stepanauskas, R. Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system. Frontiers in Microbiology, 10, (2019): 1262, doi:10.3389/fmicb.2019.01262., Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds., This work was supported by the U.S. National Science Foundation’s Dimensions of Biodiversity Program [OCE-1136488 (to RS), OCE-1136727 (to SMS) and OCE-1136451 (to CV)], as well as DEB-1441717 and OCE-1335810 (to RS), and the DOE JGI CSP project 1477.

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 6 (2017): e18990, doi:10.7554/eLife.18990., Anaerobic thermophiles inhabit relic environments that resemble the early Earth. However, the lineage of these modern organisms co-evolved with our planet. Hence, these organisms carry both ancestral and acquired genes and serve as models to reconstruct early metabolism. Based on comparative genomic and proteomic analyses, we identified two distinct groups of genes in Thermovibrio ammonificans: the first codes for enzymes that do not require oxygen and use substrates of geothermal origin; the second appears to be a more recent acquisition, and may reflect adaptations to cope with the rise of oxygen on Earth. We propose that the ancestor of the Aquificae was originally a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid pathway for CO2 fixation. With the gradual rise of oxygen in the atmosphere, more efficient terminal electron acceptors became available and this lineage acquired genes that increased its metabolic flexibility while retaining ancestral metabolic traits., National Science Foundation (MCB 04-56676), (OCE 03-27353), (MCB 08-43678), (OCE 09-37371), (OCE 11-24141), (MCB 15-17567), (OCE-1136727); National Aeronautics and Space Administration (NNX15AM18G)

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 6 (2017): e18990, doi:10.7554/eLife.18990., Anaerobic thermophiles inhabit relic environments that resemble the early Earth. However, the lineage of these modern organisms co-evolved with our planet. Hence, these organisms carry both ancestral and acquired genes and serve as models to reconstruct early metabolism. Based on comparative genomic and proteomic analyses, we identified two distinct groups of genes in Thermovibrio ammonificans: the first codes for enzymes that do not require oxygen and use substrates of geothermal origin; the second appears to be a more recent acquisition, and may reflect adaptations to cope with the rise of oxygen on Earth. We propose that the ancestor of the Aquificae was originally a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid pathway for CO2 fixation. With the gradual rise of oxygen in the atmosphere, more efficient terminal electron acceptors became available and this lineage acquired genes that increased its metabolic flexibility while retaining ancestral metabolic traits., National Science Foundation (MCB 04-56676), (OCE 03-27353), (MCB 08-43678), (OCE 09-37371), (OCE 11-24141), (MCB 15-17567), (OCE-1136727); National Aeronautics and Space Administration (NNX15AM18G)

Hydrothermal vent systems are inhabited by dense benthic communities adapted to extreme conditions such as high temperature, hydrogen sulphide (H2S) and elevated fluxes of metals. In the present work, a wide range of trace elements (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sb, Se, V and Zn) were measured in tissues of three tube dwelling annelids, Alvinella pompejana, Alvinella caudata and Riftia pachyptila, which colonize distinct habitats of the East Pacific Rise (EPR) at 2500 m depth. Metals concentrations in alvinellids were often 2–4 orders of magnitude higher than those commonly found in marine organisms, while much lower values were observed in the vestimentiferan polychaete. Mobility of trace elements was further characterized in tissues of A. pompejana where metals appeared mostly in insoluble forms, i.e. associated with hydrated oxides and sulphides. Arsenic was mainly present in a weakly insoluble form and with concentrations in the branchial tentacles of alvinellids, approximately 5–15 fold higher than those measured in the thorax. Chemical speciation of this element in tissues of the three polychaete species revealed a major contribution of methylated arsenic compounds, like dimethylarsinate (DMA) and, to a lower extent, monomethylarsonate (MMA) and trimethylarsine oxide (TMAO). Although the biotransformation of inorganic arsenic might represent a detoxification mechanism in polychaetes from hydrothermal vents, the elevated levels of methylated forms of arsenic in branchial tissues also suggest an ecological role of this element as an antipredatory strategy for more vulnerable tissues toward generalist consumers.

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 941, doi:10.3389/fmicb.2016.00941., Pockmarks are crater-like depression on the seafloor associated with hydrocarbon ascent through muddy sediments in continental shelves around the world. In this study, we examine the diversity and distribution of benthic microbial communities at shallow-water pockmarks adjacent to the Middle Adriatic Ridge. We integrate microbial diversity data with characterization of local hydrocarbons concentrations and sediment geochemistry. Our results suggest these pockmarks are enriched in sedimentary hydrocarbons, and host a microbial community dominated by Bacteria, even in deeper sediment layers. Pockmark sediments showed higher prokaryotic abundance and biomass than surrounding sediments, potentially due to the increased availability of organic matter and higher concentrations of hydrocarbons linked to pockmark activity. Prokaryotic diversity analyses showed that the microbial communities of these shallow-water pockmarks are unique, and comprised phylotypes associated with the cycling of sulfur and nitrate compounds, as well as numerous know hydrocarbon degraders. Altogether, this study suggests that shallow-water pockmark habitats enhance the diversity of the benthic prokaryotic biosphere by providing specialized environmental niches., This work was partially supported by a Postdoctoral Fellowship from the Center for Dark Energy Biosphere Investigations [C-DEBI, grant OCE-0939564] awarded to DG, by National Science Foundation grant OCE 11-24141 to CV, and European Science Foundation EuroDeep BIOFUN grant CTM2007-28739-E to EM. This article commits to EU HERMIONE [contract no. 226354] and CoCoNet [contract no. 287844] programs, and the Italian MIUR flag Ritmare within the National Research Program 2011–2013.

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 941, doi:10.3389/fmicb.2016.00941., Pockmarks are crater-like depression on the seafloor associated with hydrocarbon ascent through muddy sediments in continental shelves around the world. In this study, we examine the diversity and distribution of benthic microbial communities at shallow-water pockmarks adjacent to the Middle Adriatic Ridge. We integrate microbial diversity data with characterization of local hydrocarbons concentrations and sediment geochemistry. Our results suggest these pockmarks are enriched in sedimentary hydrocarbons, and host a microbial community dominated by Bacteria, even in deeper sediment layers. Pockmark sediments showed higher prokaryotic abundance and biomass than surrounding sediments, potentially due to the increased availability of organic matter and higher concentrations of hydrocarbons linked to pockmark activity. Prokaryotic diversity analyses showed that the microbial communities of these shallow-water pockmarks are unique, and comprised phylotypes associated with the cycling of sulfur and nitrate compounds, as well as numerous know hydrocarbon degraders. Altogether, this study suggests that shallow-water pockmark habitats enhance the diversity of the benthic prokaryotic biosphere by providing specialized environmental niches., This work was partially supported by a Postdoctoral Fellowship from the Center for Dark Energy Biosphere Investigations [C-DEBI, grant OCE-0939564] awarded to DG, by National Science Foundation grant OCE 11-24141 to CV, and European Science Foundation EuroDeep BIOFUN grant CTM2007-28739-E to EM. This article commits to EU HERMIONE [contract no. 226354] and CoCoNet [contract no. 287844] programs, and the Italian MIUR flag Ritmare within the National Research Program 2011–2013.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

Author Posting. © The Oceanography Society, 2012. This article is posted here by permission of The Oceanography Society for personal use, not for redistribution. The definitive version was published in Oceanography 25, no. 1 (2012): 218–233, doi:10.5670/oceanog.2012.21., Chemolithoautotrophic microorganisms are at the nexus of hydrothermal systems by effectively transferring the energy from the geothermal source to the higher trophic levels. While the validity of this conceptual framework is well established at this point, there are still significant gaps in our understanding of the microbiology and biogeochemistry of deep-sea hydrothermal systems. Important questions in this regard are: (1) How much, at what rates, and where in the system is organic carbon being produced? (2) What are the dominant autotrophs, where do they reside, and what is the relative importance of free-swimming, biofilm-forming, and symbiotic microbes? (3) Which metabolic pathways are they using to conserve energy and to fix carbon? (4) How does community-wide gene expression in fluid and biofilm communities compare? and (5) How efficiently is the energy being utilized, transformed into biomass, and transferred to higher trophic levels? In particular, there is currently a notable lack of process-oriented studies that would allow an assessment of the larger role of these ecosystems in global biogeochemical cycles. By combining the presently available powerful "omic" and single-cell tools with thermodynamic modeling, experimental approaches, and new in situ instrumentation to measure rates and concentrations, it is now possible to bring our understanding of these truly fascinating ecosystems to a new level and to place them in a quantitative framework and thus a larger global context., This review was written with support from NSF grants OCE-1136727, OCE-1038131, and OCE-1131095 (SMS) and OCE-1136451 (CV). Research mentioned in the review that was carried out in the labs of CV and SMS was supported by NSF grants MCB-0843678 (CV) and OCE-0452333 (SMS).

Author Posting. © The Oceanography Society, 2012. This article is posted here by permission of The Oceanography Society for personal use, not for redistribution. The definitive version was published in Oceanography 25, no. 1 (2012): 218–233, doi:10.5670/oceanog.2012.21., Chemolithoautotrophic microorganisms are at the nexus of hydrothermal systems by effectively transferring the energy from the geothermal source to the higher trophic levels. While the validity of this conceptual framework is well established at this point, there are still significant gaps in our understanding of the microbiology and biogeochemistry of deep-sea hydrothermal systems. Important questions in this regard are: (1) How much, at what rates, and where in the system is organic carbon being produced? (2) What are the dominant autotrophs, where do they reside, and what is the relative importance of free-swimming, biofilm-forming, and symbiotic microbes? (3) Which metabolic pathways are they using to conserve energy and to fix carbon? (4) How does community-wide gene expression in fluid and biofilm communities compare? and (5) How efficiently is the energy being utilized, transformed into biomass, and transferred to higher trophic levels? In particular, there is currently a notable lack of process-oriented studies that would allow an assessment of the larger role of these ecosystems in global biogeochemical cycles. By combining the presently available powerful "omic" and single-cell tools with thermodynamic modeling, experimental approaches, and new in situ instrumentation to measure rates and concentrations, it is now possible to bring our understanding of these truly fascinating ecosystems to a new level and to place them in a quantitative framework and thus a larger global context., This review was written with support from NSF grants OCE-1136727, OCE-1038131, and OCE-1131095 (SMS) and OCE-1136451 (CV). Research mentioned in the review that was carried out in the labs of CV and SMS was supported by NSF grants MCB-0843678 (CV) and OCE-0452333 (SMS).

Author Posting. © National Shellfisheries Association, 2008. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 27 (2008): 177-190, doi:10.2983/0730-8000(2008)27[177:IBVFCT]2.0.CO;2., In April 1991, submarine volcanic eruptions initiated the formation of numerous hydrothermal vents between 9°45′ and 9°52′N along the crest of the East Pacific Rise (EPR). Dramatic changes in biological community structure and vent fluid chemistry have been documented throughout this region since the eruptive event. By April 2004, mussels (Bathymodiolus thermophilus) dominated the faunal assemblages at several of the vent sites formed during of after the 1991 eruptions, whereas other habitats within the region were dominated by the vestimentiferan Riftia pachyptila. In the present paper, we build upon the extensive data sets obtained at these sites over the past decade and describe a manipulative experiment (conducted at 9°49.94′N; 104°14.43′W on the EPR) designed to assess interrelationships between vent fluid chemistry, temperature, biological community structure, and seismic activity. To this end, in situ voltammetric systems and thermal probes were used to measure H2S/HS− and temperature over time in a denuded region of an extensive mussel bed in which an exclusion cage was placed to inhibit the subsequent migration of mussels into the denuded area. Fluid samples were taken from the same locations to characterize the associated microbial constituents. Basalt blocks, which were placed in the cage in April 2004 and subsequently recovered in April 2005, were colonized by more than 25 different species of invertebrates, including numerous vestimentiferans and remarkably few mussels. Recorded temporal changes in vent fluid chemistry and temperature regimes, when coupled with microbiological characterization of the vent fluids and seismic activity data obtained from ocean bottom seismometers, shed considerable light on factors controlling biological community structure in these hydrothermal ecosystems., Supported by NSF Grants OCE-9529819, ESI-0087679 (RAL), OCE-0327353 (RAL and CV), OCE-0327261, OCE-0451983 (TS), MCB-0456676, CHE-0221978 (CV), OCE-0326434 (GWL), and OCE-0327283 (MT), the Deep Ocean Exploration Institute at the Woods Hole Oceanographic Institution, and the New Jersey Agricultural Experiment Station at Rutgers University.

Author Posting. © National Shellfisheries Association, 2008. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 27 (2008): 177-190, doi:10.2983/0730-8000(2008)27[177:IBVFCT]2.0.CO;2., In April 1991, submarine volcanic eruptions initiated the formation of numerous hydrothermal vents between 9°45′ and 9°52′N along the crest of the East Pacific Rise (EPR). Dramatic changes in biological community structure and vent fluid chemistry have been documented throughout this region since the eruptive event. By April 2004, mussels (Bathymodiolus thermophilus) dominated the faunal assemblages at several of the vent sites formed during of after the 1991 eruptions, whereas other habitats within the region were dominated by the vestimentiferan Riftia pachyptila. In the present paper, we build upon the extensive data sets obtained at these sites over the past decade and describe a manipulative experiment (conducted at 9°49.94′N; 104°14.43′W on the EPR) designed to assess interrelationships between vent fluid chemistry, temperature, biological community structure, and seismic activity. To this end, in situ voltammetric systems and thermal probes were used to measure H2S/HS− and temperature over time in a denuded region of an extensive mussel bed in which an exclusion cage was placed to inhibit the subsequent migration of mussels into the denuded area. Fluid samples were taken from the same locations to characterize the associated microbial constituents. Basalt blocks, which were placed in the cage in April 2004 and subsequently recovered in April 2005, were colonized by more than 25 different species of invertebrates, including numerous vestimentiferans and remarkably few mussels. Recorded temporal changes in vent fluid chemistry and temperature regimes, when coupled with microbiological characterization of the vent fluids and seismic activity data obtained from ocean bottom seismometers, shed considerable light on factors controlling biological community structure in these hydrothermal ecosystems., Supported by NSF Grants OCE-9529819, ESI-0087679 (RAL), OCE-0327353 (RAL and CV), OCE-0327261, OCE-0451983 (TS), MCB-0456676, CHE-0221978 (CV), OCE-0326434 (GWL), and OCE-0327283 (MT), the Deep Ocean Exploration Institute at the Woods Hole Oceanographic Institution, and the New Jersey Agricultural Experiment Station at Rutgers University.

Author Posting. © Springer, 2008. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Extremophiles 12 (2008): 627-640, doi:10.1007/s00792-008-0167-5., 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., This research was supported by NSF grants MCB 04-56676 (C.V.), OCE 03-27353 (C.V.), MCB 04-56689 (S.M.S.), a grant from the New Jersey Agricultural Experiment Station to C.V., and a NIH Ph.D. Training Program in Biotechnology Fellowship (NIH NIGMS 5 T32 GM08339) to J.V. M.H. was supported through a postdoctoral scholarship from the Woods Hole Oceanographic Institution.

Author Posting. © Springer, 2008. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Extremophiles 12 (2008): 627-640, doi:10.1007/s00792-008-0167-5., 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., This research was supported by NSF grants MCB 04-56676 (C.V.), OCE 03-27353 (C.V.), MCB 04-56689 (S.M.S.), a grant from the New Jersey Agricultural Experiment Station to C.V., and a NIH Ph.D. Training Program in Biotechnology Fellowship (NIH NIGMS 5 T32 GM08339) to J.V. M.H. was supported through a postdoctoral scholarship from the Woods Hole Oceanographic Institution.

We report new in situ observations and laboratory studies of specimens of a small (diameter 2.4–7.5 cm) strikingly hexagonal form originally described from sedimented steps in a wall of the axial valley of the Mid-Atlantic Ridge (water depth 3430–3575 m) near 26°N, 45°W that appears to be identical to the iconic form Paleodictyon nodosum described as a trace fossil from Eocene flysch deposits at sites in Europe and Wales. Our findings follow: • The form is apparently agglutinated in sea floor sediment (a veneer of calcareous lutite over red metalliferous sediment) and consists of three equidistant rows of tiny holes (diameter 1 mm) that intersect at an angle of 120° and continuously connect through vertical shafts (length 2–3 mm) with an underlying horizontal network of tubes or tunnels identical with the fossil form. • The number of rows of holes and spacing of rows increase with overall diameter of the form indicative of organic growth. • The form is shaped like a shield surrounded by a lip and moat with surface relief (0.5 cm) that is absent in the fossil form. The surface relief exposes the underlying red sediment and may have been produced either by excavation (constructional origin) or by infaunal growth (body form). • Protoplasm is absent in recovered specimens, as indicated by negative results of staining techniques, explained by either initial absence or loss. • Genetic sequencing of material from the form identified different foraminifera that had settled on the pattern of holes which acts as a baffle to trap organic matter. • Models in flume tanks show that the shield-like form deflects flow of ocean currents into a self-ventilating structure capable of aerating and of circulating organic particles through the tubes or tunnels. • Microbial counting techniques indicated background abundances within and outside the form. We come to two alternative interpretations of the findings resolvable with further studies: • The modern P. nodosum is a burrow consistent

We report new in situ observations and laboratory studies of specimens of a small (diameter 2.4–7.5 cm) strikingly hexagonal form originally described from sedimented steps in a wall of the axial valley of the Mid-Atlantic Ridge (water depth 3430–3575 m) near 26°N, 45°W that appears to be identical to the iconic form Paleodictyon nodosum described as a trace fossil from Eocene flysch deposits at sites in Europe and Wales. Our findings follow: • The form is apparently agglutinated in sea floor sediment (a veneer of calcareous lutite over red metalliferous sediment) and consists of three equidistant rows of tiny holes (diameter 1 mm) that intersect at an angle of 120° and continuously connect through vertical shafts (length 2–3 mm) with an underlying horizontal network of tubes or tunnels identical with the fossil form. • The number of rows of holes and spacing of rows increase with overall diameter of the form indicative of organic growth. • The form is shaped like a shield surrounded by a lip and moat with surface relief (0.5 cm) that is absent in the fossil form. The surface relief exposes the underlying red sediment and may have been produced either by excavation (constructional origin) or by infaunal growth (body form). • Protoplasm is absent in recovered specimens, as indicated by negative results of staining techniques, explained by either initial absence or loss. • Genetic sequencing of material from the form identified different foraminifera that had settled on the pattern of holes which acts as a baffle to trap organic matter. • Models in flume tanks show that the shield-like form deflects flow of ocean currents into a self-ventilating structure capable of aerating and of circulating organic particles through the tubes or tunnels. • Microbial counting techniques indicated background abundances within and outside the form. We come to two alternative interpretations of the findings resolvable with further studies: • The modern P. nodosum is a burrow consistent

Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO2 fixation. This is the first time that strains of the Hydrogenothermaceae and ‘Desulfurobacteriaceae’ have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and ‘Desulfurobacteriaceae’ use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.

Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO2 fixation. This is the first time that strains of the Hydrogenothermaceae and ‘Desulfurobacteriaceae’ have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and ‘Desulfurobacteriaceae’ use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.

Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO2 fixation. This is the first time that strains of the Hydrogenothermaceae and ‘Desulfurobacteriaceae’ have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and ‘Desulfurobacteriaceae’ use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.