Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 103 cells ml–1) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to ‘omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis.
Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus lineages and their transcripts were more abundant than others in individual vent sites, indicating differential fitness among lineages. The relative abundances of lineages represented by SAGs in each of the samples matched phylogenetic relationships based on single-copy universal genes, and genes related to nitrogen fixation and the CRISPR/Cas immune system were among those differentiating the clades. Lineages possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.
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.
The exploration of Earth’s terrestrial subsurface biosphere has led to the discovery of several new archaeal lineages of evolutionary significance. Similarly, the deep subseafloor crustal biosphere also harbors many unique, uncultured archaeal taxa, including those belonging to Candidatus Hydrothermarchaeota, formerly known as Marine Benthic Group-E. Recently, Hydrothermarchaeota was identified as an abundant lineage of Juan de Fuca Ridge flank crustal fluids, suggesting its adaptation to this extreme environment. Through the investigation of single-cell and metagenome-assembled genomes, we provide insight into the lineage’s evolutionary history and metabolic potential. Phylogenomic analysis reveals the Hydrothermarchaeota to be an early-branching archaeal phylum, branching between the superphylum DPANN, Euryarchaeota, and Asgard lineages. Hydrothermarchaeota genomes suggest a potential for dissimilative and assimilative carbon monoxide oxidation (carboxydotrophy), as well as sulfate and nitrate reduction. There is also a prevalence of chemotaxis and motility genes, indicating adaptive strategies for this nutrient-limited fluid-rock environment. These findings provide the first genomic interpretations of the Hydrothermarchaeota phylum and highlight the anoxic, hot, deep marine crustal biosphere as an important habitat for understanding the evolution of early life.
It is generally accepted that diverse, poorly characterized microorganisms reside deep within Earth’s crust. One such lineage of deep subsurface-dwelling bacteria is an uncultivated member of the Firmicutes phylum that can dominate molecular surveys from both marine and continental rock fracture fluids, sometimes forming the sole member of a single-species microbiome. Here, we reconstructed a genome from basalt-hosted fluids of the deep subseafloor along the eastern Juan de Fuca Ridge flank and used a phylogenomic analysis to show that, despite vast differences in geographic origin and habitat, it forms a monophyletic clade with the terrestrial deep subsurface genome of “Candidatus Desulforudis audaxviator” MP104C. While a limited number of differences were observed between the marine genome of “Candidatus Desulfopertinax cowenii” modA32 and its terrestrial relative that may be of potential adaptive importance, here it is revealed that the two are remarkably similar thermophiles possessing the genetic capacity for motility, sporulation, hydrogenotrophy, chemoorganotrophy, dissimilatory sulfate reduction, and the ability to fix inorganic carbon via the Wood-Ljungdahl pathway for chemoautotrophic growth. Our results provide insights into the genetic repertoire within marine and terrestrial members of a bacterial lineage that is widespread in the global deep subsurface biosphere, and provides a natural means to investigate adaptations specific to these two environments.
| URL | https://www.bco-dmo.org/dataset/660802 |
|---|---|
| Download URL | https://www.bco-dmo.org/dataset/660802/data/download |
| Media Type | text/tab-separated-values |
| Created | October 5, 2016 |
| Modified | April 18, 2019 |
| State | Final no updates expected |
| Brief Description | Single amplified genomes (SAGs) of microbial cells isolated from Crab Spa, East Pacific Rise |
AT15-38: Samples for SAGs were obtained using the so-called ‘titanium major samplers’ (von Damm et al, 1985). Replicate samples of 1 ml aliquots of water were cryopreserved with 6% glycine betaine (Sigma) or 15% glycerol and stored at -80 ºC for the ”Single Cell” aliquot.
AT26-10: Background samples were obtained from the IGTs, in which the incubations were carried out. Just in this case, an aliquot was removed after sample retrieval and before starting the incubation. During AT26-10, samples were preserved with Gly-TE and stored at -80.
Cells were sorted, identified and sequenced by the Bigelow Laboratory Single Cell Genomics FacilityCenter (SCGC), following SCGCthe facilities’s standard practices: SCGC_Services_Description.pdf
On average, at least 5 million 2×150 bp or longer paired-end reads were generated per SAG using in-house MiSeq and NextSeq (Illumina) instruments. The obtained reads were pre-processed and, de novo, assembled and quality-controlled using algorithms SCGC’s standard protocols that are optimized for single cell MDA products . A combination of tetramer homogeneity tests and blast searches against reference databases is used to detect potential DNA contaminants among the assembled contigs. Benchmark data demonstrating SCGC SAG WGS whole genome sequencing pipeline performance is available from the SCGC website http://data.bigelow.org/~scgc/WGS_benchmark_data/.
Genome annotation was performed through IMG (http://img-stage.jgi-psf.org/cgi-bin/submit/main.cgi).
MiSeq and NextSeq (Illumina) sequencers
General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
Isobaric Gas Tight (IGT) samplers, designed and built by scientists and engineers at WHOI, are titanium instruments designed to be used with deep submergence vehicles to sample corrosive hydrothermal vent fluids at high temperature and high pressure. The IGT prevents the sampled fluid from degassing as pressure decreases during the vehicle’s ascent to the surface.
taxonomic group or entity code, such as the ten digit number from the NODC Taxonomic List, v. 8.
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
taxonomic group or entity. This may be a family, class, genus, species, etc.; usually this parameter will contain a mixture of taxonomic entities.
The origin of the sample used for the single cell sorting:
Background: Cell sorted from the natural water samples
Control: Cell sorted from the natural water samples incubated in isobaric chambers without any amendments
NO3-/H2, ~24C: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of NO3- (nitrate) and hydrogen (H2)
H2 only: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of hydrogen (H2)
NO3- only: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of NO3- (nitrate)hydrogen (H2)
O2, ~110uM: Cell sorted from the natural water samples incubated at 24oC in isobaric chambers with the addition of 110uM of O2 (oxygen)
NO3-/H2, ~50C: Cell sorted from the natural water samples incubated at 50oC in isobaric chambers with the addition of NO3- (nitrate) and hydrogen (H2)
Experimental conditions applied to experimental units. In comparative experiments, members of the complementary group, the control group, receive either no treatment or a standard treatment.
Method used to collect sample.
latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes
longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes
Link to an external data entry.
| Name | Affiliation | Contact |
|---|---|---|
| Ramunas Stepanauskas | Bigelow Laboratory for Ocean Sciences | ✓ |
| Nancy Copley | Bigelow Laboratory for Ocean Sciences | ✓ |
| Project Title | An Integrated Study of Energy Metabolism, Carbon Fixation, and Colonization Mechanisms in Chemosynthetic Microbial Communities at Deep-Sea Vents |
|---|---|
| Acronym | Microbial Communities at Deep-Sea Vents |
| URL | https://www.bco-dmo.org/project/2216 |
| Created | June 11, 2012 |
| Modified | June 11, 2012 |
Deep-sea hydrothermal vents, first discovered in 1977, are poster child ecosystems where microbial chemosynthesis rather than photosynthesis is the primary source of organic carbon. Significant gaps remain in our understanding of the underlying microbiology and biogeochemistry of these fascinating ecosystems. Missing are the identification of specific microorganisms mediating critical reactions in various geothermal systems, metabolic pathways used by the microbes, rates of the catalyzed reactions, amounts of organic carbon being produced, and the larger role of these ecosystems in global biogeochemical cycles. To fill these gaps, the investigators will conduct a 3-year interdisciplinary, international hypothesis-driven research program to understand microbial processes and their quantitative importance at deep-sea vents. Specifically, the investigators will address the following objectives: 1. Determine key relationships between the taxonomic, genetic and functional diversity, as well as the mechanisms of energy and carbon transfer, in deep-sea hydrothermal vent microbial communities. 2. Identify the predominant metabolic pathways and thus the main energy sources driving chemoautotrophic production in high and low temperature diffuse flow vents. 3. Determine energy conservation efficiency and rates of aerobic and anaerobic chemosynthetic primary productivity in high and low temperature diffuse flow vents. 4. Determine gene expression patterns in diffuse-flow vent microbial communities during attachment to substrates and the development of biofilms.
Integration: To address these objectives and to characterize the complexity of microbially-catalyzed processes at deep-sea vents at a qualitatively new level, we will pursue an integrated approach that couples an assessment of taxonomic diversity using cultivation-dependent and -independent approaches with methodologies that address genetic diversity, including a) metagenomics (genetic potential and diversity of community), b) single cell genomics (genetic potential and diversity of uncultured single cells), c) meta-transcriptomics and -proteomics (identification and function of active community members, realized potential of the community). To assess function and response to the environment, these approaches will be combined with 1) measurement of in situ rates of chemoautotrophic production, 2) geochemical characterization of microbial habitats, and 3) shipboard incubations under simulated in situ conditions (hypothesis testing under controlled physicochemical conditions). Network approaches and mathematical simulation will be used to reconstruct the metabolic network of the natural communities. A 3-day long project meeting towards the end of the second year will take place in Woods Hole. This Data Integration and Synthesis meeting will allow for progress reports and presentations from each PI, postdoc, and/or student, with the aim of synthesizing data generated to facilitate the preparation of manuscripts.
Intellectual Merit. Combining the community expression profile with diversity and metagenomic analyses as well as process and habitat characterization will be unique to hydrothermal vent microbiology. The approach will provide new insights into the functioning of deep-sea vent microbial communities and the constraints regulating the interactions between the microbes and their abiotic and biotic environment, ultimately enabling us to put these systems into a quantitative framework and thus a larger global context.
Broader Impacts. This is an interdisciplinary and collaborative effort between 4 US and 4 foreign institutions, creating unique opportunities for networking and fostering international collaborations. This will also benefit the involved students (2 graduate, several undergraduate) and 2 postdoctoral associates. This project will directly contribute to many educational and public outreach activities of the involved PIs, including the WHOI Dive & Discover program; single cell genomics workshops and Cafe Scientifique (Bigelow); REU (WHOI, Bigelow, CIW); COSEE and RIOS (Rutgers), and others. The proposed research fits with the focus of a number of multidisciplinary and international initiatives, in which PIs are active members (SCOR working group on Hydrothermal energy and the ocean carbon cycle, http://www.scorint. org/Working_Groups/wg135.htm; Deep Carbon Observatory at CIW, https://dco.gl.ciw.edu/; Global Biogeochemical Flux (GBF) component of the Ocean Observatories Initiative (OOI), http://www.whoi.edu/GBF-OOI/page.do?pid=41475)
| Name | Affiliation | Role |
|---|---|---|
| Stefan M. Sievert | Woods Hole Oceanographic Institution (WHOI) | Lead Principal Investigator |
| Costantino Vetriani | Rutgers University | Principal Investigator |
| Dionysis I. Foustoukos | Carnegie Institution for Science (CIS) | Principal Investigator |
| Ramunas Stepanauskas | Bigelow Laboratory for Ocean Sciences | Principal Investigator |
| Craig Taylor | Woods Hole Oceanographic Institution (WHOI) | Co-Principal Investigator |
| Jeffrey S. Seewald | Max Planck Institute for Marine Microbiology (MPI) | International Collaborator |
| Nadine Le Bris | University of Greifswald | International Collaborator |
| Niculina Musat | Shanghai Jiao Tong University (SJTU) | International Collaborator |
| Thomas Schweder | Laboratoire d'Écogéochimie des Environnements Benthiques (LECOB) | International Collaborator |
| Fengping Wang | Woods Hole Oceanographic Institution (WHOI) | Co-Principal Investigator |
| Project Title | An Integrated Study of Energy Metabolism, Carbon Fixation, and Colonization Mechanisms in Chemosynthetic Microbial Communities at Deep-Sea Vents |
|---|---|
| Acronym | Microbial Communities at Deep-Sea Vents |
| URL | https://www.bco-dmo.org/project/2216 |
| Created | June 11, 2012 |
| Modified | June 11, 2012 |
Deep-sea hydrothermal vents, first discovered in 1977, are poster child ecosystems where microbial chemosynthesis rather than photosynthesis is the primary source of organic carbon. Significant gaps remain in our understanding of the underlying microbiology and biogeochemistry of these fascinating ecosystems. Missing are the identification of specific microorganisms mediating critical reactions in various geothermal systems, metabolic pathways used by the microbes, rates of the catalyzed reactions, amounts of organic carbon being produced, and the larger role of these ecosystems in global biogeochemical cycles. To fill these gaps, the investigators will conduct a 3-year interdisciplinary, international hypothesis-driven research program to understand microbial processes and their quantitative importance at deep-sea vents. Specifically, the investigators will address the following objectives: 1. Determine key relationships between the taxonomic, genetic and functional diversity, as well as the mechanisms of energy and carbon transfer, in deep-sea hydrothermal vent microbial communities. 2. Identify the predominant metabolic pathways and thus the main energy sources driving chemoautotrophic production in high and low temperature diffuse flow vents. 3. Determine energy conservation efficiency and rates of aerobic and anaerobic chemosynthetic primary productivity in high and low temperature diffuse flow vents. 4. Determine gene expression patterns in diffuse-flow vent microbial communities during attachment to substrates and the development of biofilms.
Integration: To address these objectives and to characterize the complexity of microbially-catalyzed processes at deep-sea vents at a qualitatively new level, we will pursue an integrated approach that couples an assessment of taxonomic diversity using cultivation-dependent and -independent approaches with methodologies that address genetic diversity, including a) metagenomics (genetic potential and diversity of community), b) single cell genomics (genetic potential and diversity of uncultured single cells), c) meta-transcriptomics and -proteomics (identification and function of active community members, realized potential of the community). To assess function and response to the environment, these approaches will be combined with 1) measurement of in situ rates of chemoautotrophic production, 2) geochemical characterization of microbial habitats, and 3) shipboard incubations under simulated in situ conditions (hypothesis testing under controlled physicochemical conditions). Network approaches and mathematical simulation will be used to reconstruct the metabolic network of the natural communities. A 3-day long project meeting towards the end of the second year will take place in Woods Hole. This Data Integration and Synthesis meeting will allow for progress reports and presentations from each PI, postdoc, and/or student, with the aim of synthesizing data generated to facilitate the preparation of manuscripts.
Intellectual Merit. Combining the community expression profile with diversity and metagenomic analyses as well as process and habitat characterization will be unique to hydrothermal vent microbiology. The approach will provide new insights into the functioning of deep-sea vent microbial communities and the constraints regulating the interactions between the microbes and their abiotic and biotic environment, ultimately enabling us to put these systems into a quantitative framework and thus a larger global context.
Broader Impacts. This is an interdisciplinary and collaborative effort between 4 US and 4 foreign institutions, creating unique opportunities for networking and fostering international collaborations. This will also benefit the involved students (2 graduate, several undergraduate) and 2 postdoctoral associates. This project will directly contribute to many educational and public outreach activities of the involved PIs, including the WHOI Dive & Discover program; single cell genomics workshops and Cafe Scientifique (Bigelow); REU (WHOI, Bigelow, CIW); COSEE and RIOS (Rutgers), and others. The proposed research fits with the focus of a number of multidisciplinary and international initiatives, in which PIs are active members (SCOR working group on Hydrothermal energy and the ocean carbon cycle, http://www.scorint. org/Working_Groups/wg135.htm; Deep Carbon Observatory at CIW, https://dco.gl.ciw.edu/; Global Biogeochemical Flux (GBF) component of the Ocean Observatories Initiative (OOI), http://www.whoi.edu/GBF-OOI/page.do?pid=41475)
| Name | Affiliation | Role |
|---|---|---|
| Stefan M. Sievert | Woods Hole Oceanographic Institution (WHOI) | Lead Principal Investigator |
| Costantino Vetriani | Rutgers University | Principal Investigator |
| Dionysis I. Foustoukos | Carnegie Institution for Science (CIS) | Principal Investigator |
| Ramunas Stepanauskas | Bigelow Laboratory for Ocean Sciences | Principal Investigator |
| Craig Taylor | Woods Hole Oceanographic Institution (WHOI) | Co-Principal Investigator |
| Jeffrey S. Seewald | Max Planck Institute for Marine Microbiology (MPI) | International Collaborator |
| Nadine Le Bris | University of Greifswald | International Collaborator |
| Niculina Musat | Shanghai Jiao Tong University (SJTU) | International Collaborator |
| Thomas Schweder | Laboratoire d'Écogéochimie des Environnements Benthiques (LECOB) | International Collaborator |
| Fengping Wang | Woods Hole Oceanographic Institution (WHOI) | Co-Principal Investigator |
Half of the microbial cells in the Earth’s oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.
