Life strategies for Aminicenantia in subseafloor oceanic crust.

After decades studying the microbial "deep biosphere" in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use "primordial" siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.

Structure-informed microbial population genetics elucidate selective pressures that shape protein evolution.

Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, resolving adaptive, neutral, or purifying processes of evolution from intrapopulation genomic variation remains a challenge, partly due to the sole reliance on gene sequences to interpret variants. Here, we describe an approach to analyze genetic variation in the context of predicted protein structures and apply it to a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between genetic variation and protein structure. In a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand-binding sites as a function of nitrate concentrations, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our work yields insights into the governing principles of evolution and enables structure-aware investigations of microbial population genetics.

Spatial and temporal dynamics of SAR11 marine bacteria across a nearshore to offshore transect in the tropical Pacific Ocean.

Surveys of microbial communities across transitions coupled with contextual measures of the environment provide a useful approach to dissect the factors determining distributions of microorganisms across ecological niches. Here, monthly time-series samples of surface seawater along a transect spanning the nearshore coastal environment within Kane'ohe Bay on the island of O'ahu, Hawai'i, and the adjacent offshore environment were collected to investigate the diversity and abundance of SAR11 marine bacteria (order Pelagibacterales) over a 2-year time period. Using 16S ribosomal RNA gene amplicon sequencing, the spatiotemporal distributions of major SAR11 subclades and exact amplicon sequence variants (ASVs) were evaluated. Seven of eight SAR11 subclades detected in this study showed distinct subclade distributions across the coastal to offshore environments. The SAR11 community was dominated by seven (of 106 total) SAR11 ASVs that made up an average of 77% of total SAR11. These seven ASVs spanned five different SAR11 subclades (Ia, Ib, IIa, IV, and Va), and were recovered from all samples collected from either the coastal environment, the offshore, or both. SAR11 ASVs were more often restricted spatially to coastal or offshore environments (64 of 106 ASVs) than they were shared among coastal, transition, and offshore environments (39 of 106 ASVs). Overall, offshore SAR11 communities contained a higher diversity of SAR11 ASVs than their nearshore counterparts, with the highest diversity within the little-studied subclade IIa. This study reveals ecological differentiation of SAR11 marine bacteria across a short physiochemical gradient, further increasing our understanding of how SAR11 genetic diversity partitions into distinct ecological units.

Insights into the Cultured Bacterial Fraction of Corals.

Bacteria associated with coral hosts are diverse and abundant, with recent studies suggesting involvement of these symbionts in host resilience to anthropogenic stress. Despite their putative importance, the work dedicated to culturing coral-associated bacteria has received little attention. Combining published and unpublished data, here we report a comprehensive overview of the diversity and function of culturable bacteria isolated from corals originating from tropical, temperate, and cold-water habitats. A total of 3,055 isolates from 52 studies were considered by our metasurvey. Of these, 1,045 had full-length 16S rRNA gene sequences, spanning 138 formally described and 12 putatively novel bacterial genera across the Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria phyla. We performed comparative genomic analysis using the available genomes of 74 strains and identified potential signatures of beneficial bacterium-coral symbioses among the strains. Our analysis revealed >400 biosynthetic gene clusters that underlie the biosynthesis of antioxidant, antimicrobial, cytotoxic, and other secondary metabolites. Moreover, we uncovered genomic features-not previously described for coral-bacterium symbioses-potentially involved in host colonization and host-symbiont recognition, antiviral defense mechanisms, and/or integrated metabolic interactions, which we suggest as novel targets for the screening of coral probiotics. Our results highlight the importance of bacterial cultures to elucidate coral holobiont functioning and guide the selection of probiotic candidates to promote coral resilience and improve holistic and customized reef restoration and rehabilitation efforts. IMPORTANCE Our paper is the first study to synthesize currently available but decentralized data of cultured microbes associated with corals. We were able to collate 3,055 isolates across a number of published studies and unpublished collections from various laboratories and researchers around the world. This equated to 1,045 individual isolates which had full-length 16S rRNA gene sequences, after filtering of the original 3,055. We also explored which of these had genomes available. Originally, only 36 were available, and as part of this study, we added a further 38-equating to 74 in total. From this, we investigated potential genetic signatures that may facilitate a host-associated lifestyle. Further, such a resource is an important step in the selection of probiotic candidates, which are being investigated for promoting coral resilience and potentially applied as a novel strategy in reef restoration and rehabilitation efforts. In the spirit of open access, we have ensured this collection is available to the wider research community through the web site http://isolates.reefgenomics.org/ with the hope many scientists across the globe will ask for access to these cultures for future studies.

Ecophysiology of the Cosmopolitan OM252 Bacterioplankton (Gammaproteobacteria).

Among the thousands of species that comprise marine bacterioplankton communities, most remain functionally obscure. One key cosmopolitan group in this understudied majority is the OM252 clade of Gammaproteobacteria. Although frequently found in sequence data and even previously cultured, the diversity, metabolic potential, physiology, and distribution of this clade has not been thoroughly investigated. Here, we examined these features of OM252 bacterioplankton using a newly isolated strain and genomes from publicly available databases. We demonstrated that this group constitutes a globally distributed novel genus ("Candidatus Halomarinus"), sister to Litoricola, comprising two subclades and multiple distinct species. OM252 organisms have small genomes (median, 2.21 Mbp) and are predicted obligate aerobes capable of alternating between chemoorganoheterotrophic and chemolithotrophic growth using reduced sulfur compounds as electron donors. Subclade I genomes encode genes for the Calvin-Benson-Bassham cycle for carbon fixation. One representative strain of subclade I, LSUCC0096, had extensive halotolerance and a mesophilic temperature range for growth, with a maximum rate of 0.36 doublings/h at 35°C. Cells were curved rod/spirillum-shaped, ∼1.5 by 0.2 µm. Growth yield on thiosulfate as the sole electron donor under autotrophic conditions was roughly one-third that of heterotrophic growth, even though calculations indicated similar Gibbs energies for both catabolisms. These phenotypic data show that some "Ca. Halomarinus" organisms can switch between serving as carbon sources or sinks and indicate the likely anabolic cost of lithoautotrophic growth. Our results thus provide new hypotheses about the roles of these organisms in global biogeochemical cycling of carbon and sulfur. IMPORTANCE Marine microbial communities are teeming with understudied taxa due to the sheer numbers of species in any given sample of seawater. One group, the OM252 clade of Gammaproteobacteria, has been identified in gene surveys from myriad locations, and one isolated organism has even been genome sequenced (HIMB30). However, further study of these organisms has not occurred. Using another isolated representative (strain LSUCC0096) and publicly available genome sequences from metagenomic and single-cell genomic data sets, we examined the diversity within the OM252 clade and the distribution of these taxa in the world's oceans, reconstructed the predicted metabolism of the group, and quantified growth dynamics in LSUCC0096. Our results generate new knowledge about the previously enigmatic OM252 clade and point toward the importance of facultative chemolithoautotrophy for supporting some clades of ostensibly "heterotrophic" taxa.

Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater.

While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater environmental sample were isolated by both approaches.IMPORTANCE High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.

Sampling of basement fluids via Circulation Obviation Retrofit Kits (CORKs) for dissolved gases, fluid fixation at the seafloor, and the characterization of organic carbon.

The advanced instrumented GeoMICROBE sleds (Cowen et al., 2012) facilitate the collection of hydrothermal fluids and suspended particles in the subseafloor (basaltic) basement through Circulation Obviation Retrofit Kits (CORKs) installed within boreholes of the Integrated Ocean Drilling Program. The main components of the GeoMICROBE can be converted into a mobile pumping system (MPS) that is installed on the front basket of a submersible or remotely-operated-vehicle (ROV). Here, we provide details of a hydrothermal fluid-trap used on the MPS, through which a gastight sampler can withdraw fluids. We also applied the MPS to demonstrate the value of fixing samples at the seafloor in order to determine redox-sensitive dissolved iron concentrations and speciation measurements. To make the best use of the GeoMICROBE sleds, we describe a miniature and mobile version of the GeoMICROBE sled, which permits rapid turn-over and is relatively easy for preparation and operation. Similar to GeoMICROBE sleds, the Mobile GeoMICROBE (MGM) is capable of collecting fluid samples, filtration of suspended particles, and extraction of organics. We validate this approach by demonstrating the seafloor extraction of hydrophobic organics from a large volume (247L) of hydrothermal fluids.•We describe the design of a hydrothermal fluid-trap for use with a gastight sampler, as well as the use of seafloor fixation, through ROV- or submersible assisted mobile pumping systems.•We describe the design of a Mobile GeoMICROBE (MGM) that enhances large volume hydrothermal fluid sampling, suspended particle filtration, and organic matter extraction on the seafloor.•We provide an example of organic matter extracted and characterized from hydrothermal fluids via a MGM.

Metabolic strategies of marine subseafloor Chloroflexi inferred from genome reconstructions.

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood-Ljungdahl pathway, electron bifurcation, and ferredoxin-dependent transport-coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO2 reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H2 consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.

Single-amino acid variants reveal evolutionary processes that shape the biogeography of a global SAR11 subclade.

Members of the SAR11 order Pelagibacterales dominate the surface oceans. Their extensive diversity challenges emerging operational boundaries defined for microbial 'species' and complicates efforts of population genetics to study their evolution. Here, we employed single-amino acid variants (SAAVs) to investigate ecological and evolutionary forces that maintain the genomic heterogeneity within ubiquitous SAR11 populations we accessed through metagenomic read recruitment using a single isolate genome. Integrating amino acid and protein biochemistry with metagenomics revealed that systematic purifying selection against deleterious variants governs non-synonymous variation among very closely related populations of SAR11. SAAVs partitioned metagenomes into two main groups matching large-scale oceanic current temperatures, and six finer proteotypes that connect distant oceanic regions. These findings suggest that environmentally-mediated selection plays a critical role in the journey of cosmopolitan surface ocean microbial populations, and the idea 'everything is everywhere but the environment selects' has credence even at the finest resolutions.

Elemental Composition, Phosphorous Uptake, and Characteristics of Growth of a SAR11 Strain in Batch and Continuous Culture.

In this study, a strain of SAR11 subgroup IIIa (termed HIMB114) was grown in seawater-based batch and continuous culture in order to quantify cellular features and metabolism relevant to SAR11 ecology. We report some of the first direct measurements of cellular elemental quotas for nitrogen (N) and phosphorus (P) for SAR11, grown in batch culture: 1.4 ± 0.9 fg N and 0.44 ± 0.01 fg P, respectively, that were consistent with the small size of HIMB114 cells (average volume of 0.09 µm3). However, the mean carbon (C) cellular quota of 50 ± 47 fg C was anomalously high, but variable. The rates of phosphate (PO4 3-) uptake measured from both batch and continuous cultures were exceptionally slow: in chemostats growing at 0.3 day-1, HIMB114 took up 1.1 ± 0.3 amol P cell-1 day-1, suggesting that <30% of the cellular P requirement of HIMB114 was met by PO4 3- assimilation. The mean rate of leucine incorporation, a measure of bacterial production, during late-log-phase growth of batch HIMB114 cultures was 0.042 ± 0.02 amol Leu cell-1 h-1 While only weakly correlated with changes in specific growth rates, the onset of stationary phase resulted in decreases in cell-specific leucine incorporation that were proportional to changes in growth rate. The rates of cellular production, respiratory oxygen consumption, and changes in total organic C concentrations constrained cellular growth efficiencies to 13% ± 4%. Hence, despite a small genome and diminutively sized cells, SAR11 strain HIMB114 appears to grow at efficiencies similar to those of naturally occurring bacterioplankton communities.IMPORTANCE While SAR11 bacteria contribute a significant fraction to the total picoplankton biomass in the ocean and likely are major players in organic C and nutrient cycling, the cellular characteristics and metabolic features of most lineages have either only been hypothesized from genomes or otherwise not measured in controlled laboratory experimentation. The dearth of data on even the most basic characteristics for what is arguably the most abundant heterotroph in seawater has limited the specific consideration of SAR11 in ocean ecosystem modeling efforts. In this study, we provide measures of cellular P, N, and C, aerobic respiration, and bacterial production for a SAR11 strain growing in natural seawater medium that can be used to directly relate these features of SAR11 to biogeochemical cycling in the oceans. Through the development of a chemostat system to measure nutrient uptake during steady-state growth, we have also documented inorganic P uptake rates that allude to the importance of organic phosphorous to meet cellular P demands, even in the presence of nonlimiting PO4 3- concentrations.

Carboxydotrophy potential of uncultivated Hydrothermarchaeota from the subseafloor crustal biosphere.

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.

Divergent methyl-coenzyme M reductase genes in a deep-subseafloor Archaeoglobi.

The methyl-coenzyme M reductase (MCR) complex is a key enzyme in archaeal methane generation and has recently been proposed to also be involved in the oxidation of short-chain hydrocarbons including methane, butane, and potentially propane. The number of archaeal clades encoding the MCR continues to grow, suggesting that this complex was inherited from an ancient ancestor, or has undergone extensive horizontal gene transfer. Expanding the representation of MCR-encoding lineages through metagenomic approaches will help resolve the evolutionary history of this complex. Here, a near-complete Archaeoglobi metagenome-assembled genome (MAG; Ca. Polytropus marinifundus gen. nov. sp. nov.) was recovered from the deep subseafloor along the Juan de Fuca Ridge flank that encodes two divergent McrABG operons similar to those found in Ca. Bathyarchaeota and Ca. Syntrophoarchaeum MAGs. Ca. P. marinifundus is basal to members of the class Archaeoglobi, and encodes the genes for ß-oxidation, potentially allowing an alkanotrophic metabolism similar to that proposed for Ca. Syntrophoarchaeum. Ca. P. marinifundus also encodes a respiratory electron transport chain that can potentially utilize nitrate, iron, and sulfur compounds as electron acceptors. Phylogenetic analysis suggests that the Ca. P. marinifundus MCR operons were horizontally transferred, changing our understanding of the evolution and distribution of this complex in the Archaea.

The importance of designating type material for uncultured taxa.

Naming of uncultured Bacteria and Archaea is often inconsistent with the International Code of Nomenclature of Prokaryotes. The recent practice of proposing names for higher taxa without designation of lower ranks and nomenclature types is one of the most important inconsistencies that needs to be addressed to avoid nomenclatural instability. The Code requires names of higher taxa up to the rank of class to be derived from the type genus name, with a proposal pending to formalise this requirement for the rank of phylum. Designation of nomenclature types is crucial for providing priority to names and ensures their uniqueness and stability. However, only legitimate names proposed for axenic cultures can be used for this purpose. Candidatus names reserved for taxa lacking cultured representatives may be granted this right if recent proposals to use genome sequences as type material are endorsed, thereby allowing the Code to be fully applied to lineages represented by metagenome-assembled genomes (MAGs) or single amplified genomes (SAGs). Genome quality standards need to be considered to ensure unambiguous assignment of type material. Here, we illustrate the recommended practice by proposing nomenclature type material for four major uncultured prokaryotic lineages based on high-quality MAGs in accordance with the Code.

Author Correction: Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes.

In the version of this Article originally published, the surname of author Sandra M. McLellan was spelt incorrectly as 'MacLellan'. This has now been corrected. In addition, Fig. 2 was mistakenly duplicated in the Supplementary Information as Supplementary Fig. 2. This has now been replaced with the correct supplementary figure (shown below).

Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes.

Nitrogen fixation in the surface ocean impacts global marine nitrogen bioavailability and thus microbial primary productivity. Until now, cyanobacterial populations have been viewed as the main suppliers of bioavailable nitrogen in this habitat. Although PCR amplicon surveys targeting the nitrogenase reductase gene have revealed the existence of diverse non-cyanobacterial diazotrophic populations, subsequent quantitative PCR surveys suggest that they generally occur in low abundance. Here, we use state-of-the-art metagenomic assembly and binning strategies to recover nearly one thousand non-redundant microbial population genomes from the TARA Oceans metagenomes. Among these, we provide the first genomic evidence for non-cyanobacterial diazotrophs inhabiting surface waters of the open ocean, which correspond to lineages within the Proteobacteria and, most strikingly, the Planctomycetes. Members of the latter phylum are prevalent in aquatic systems, but have never been linked to nitrogen fixation previously. Moreover, using genome-wide quantitative read recruitment, we demonstrate that the discovered diazotrophs were not only widespread but also remarkably abundant (up to 0.3% of metagenomic reads for a single population) in both the Pacific Ocean and the Atlantic Ocean northwest. Our results extend decades of PCR-based gene surveys, and substantiate the importance of heterotrophic bacteria in the fixation of nitrogen in the surface ocean.

Expanded diversity of microbial groups that shape the dissimilatory sulfur cycle.

A critical step in the biogeochemical cycle of sulfur on Earth is microbial sulfate reduction, yet organisms from relatively few lineages have been implicated in this process. Previous studies using functional marker genes have detected abundant, novel dissimilatory sulfite reductases (DsrAB) that could confer the capacity for microbial sulfite/sulfate reduction but were not affiliated with known organisms. Thus, the identity of a significant fraction of sulfate/sulfite-reducing microbes has remained elusive. Here we report the discovery of the capacity for sulfate/sulfite reduction in the genomes of organisms from 13 bacterial and archaeal phyla, thereby more than doubling the number of microbial phyla associated with this process. Eight of the 13 newly identified groups are candidate phyla that lack isolated representatives, a finding only possible given genomes from metagenomes. Organisms from Verrucomicrobia and two candidate phyla, Candidatus Rokubacteria and Candidatus Hydrothermarchaeota, contain some of the earliest evolved dsrAB genes. The capacity for sulfite reduction has been laterally transferred in multiple events within some phyla, and a key gene potentially capable of modulating sulfur metabolism in associated cells has been acquired by putatively symbiotic bacteria. We conclude that current functional predictions based on phylogeny significantly underestimate the extent of sulfate/sulfite reduction across Earth's ecosystems. Understanding the prevalence of this capacity is integral to interpreting the carbon cycle because sulfate reduction is often coupled to turnover of buried organic carbon. Our findings expand the diversity of microbial groups associated with sulfur transformations in the environment and motivate revision of biogeochemical process models based on microbial community composition.

Corrigendum: Metagenome sequencing and 98 microbial genomes from Juan de Fuca Ridge flank subsurface fluids.

This corrects the article DOI: 10.1038/sdata.2017.37.

Genomic comparisons of a bacterial lineage that inhabits both marine and terrestrial deep subsurface systems.

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.

Viruses in the Oceanic Basement.

Microbial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 105 to 2 × 105 ml-1 (n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome.IMPORTANCE The hydrothermally active ocean basement is voluminous and likely provided conditions critical to the origins of life, but the microbiology of this vast habitat is not well understood. Viruses in particular, although integral to the origins, evolution, and ecology of all life on earth, have never been documented in basement fluids. This report provides the first estimate of free virus particles (virions) within fluids circulating through the extrusive basalt of the seafloor and describes the morphological and genetic signatures of basement viruses. These data push the known geographical limits of the virosphere deep into the ocean basement and point to a wealth of novel viral diversity, exploration of which could shed light on the early evolution of viruses.

Metagenome sequencing and 98 microbial genomes from Juan de Fuca Ridge flank subsurface fluids.

The global deep subsurface biosphere is one of the largest reservoirs for microbial life on our planet. This study takes advantage of new sampling technologies and couples them with improvements to DNA sequencing and associated informatics tools to reconstruct the genomes of uncultivated Bacteria and Archaea from fluids collected deep within the Juan de Fuca Ridge subseafloor. Here, we generated two metagenomes from borehole observatories located 311 meters apart and, using binning tools, retrieved 98 genomes from metagenomes (GFMs). Of the GFMs, 31 were estimated to be >90% complete, while an additional 17 were >70% complete. Phylogenomic analysis revealed 53 bacterial and 45 archaeal GFMs, of which nearly all were distantly related to known cultivated isolates. In the GFMs, abundant Bacteria included Chloroflexi, Nitrospirae, Acetothermia (OP1), EM3, Aminicenantes (OP8), Gammaproteobacteria, and Deltaproteobacteria, while abundant Archaea included Archaeoglobi, Bathyarchaeota (MCG), and Marine Benthic Group E (MBG-E). These data are the first GFMs reconstructed from the deep basaltic subseafloor biosphere, and provide a dataset available for further interrogation.