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Figure S1. Map of Site ODP 1244 (44°35.1784´N; 125°7.1902´W) on Hydrate Ridge, drilled on IODP Leg 204. The site is located 80 km west of
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Gas hydrates harbor gigatons of natural gas, yet their microbiomes remain understudied. We bioprospected 16S rRNA amplicons, metagenomes, and metaproteomes from methane hydrate-bearing sediments under Hydrate Ridge (offshore Oregon, USA, ODP Site 1244, 2-69 mbsf) for novel microbial metabolic and biosynthetic potential. Atribacteria sequences gener...
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Citations
... Members of the phylum Atribacterota are found in a broad range of anoxic environments, including oil reservoirs [1] and produced water [2], hot springs [3], marine sediments [4], and methane hydrates [5]. Currently, only one species, Atribacter laminatus RT761 T , has been isolated and characterized [2]. A. laminatus, a glucose fermenter, produced H 2 , acetate, and CO 2 as fermentation products and was stimulated by cocultivation with a hydrogenotrophic methanogen [2]. ...
... However, this function is not well understood due to poor cultivability and a paucity of high-quality genomes of Atribacterota. Interpretations of additional single-amplified genomes (SAGs) and metagenomeassembled genomes (MAGs) and their environmental origins have also hinted at anaerobic hydrocarbon fermentation and either syntrophic acetate oxidation or homoacetogenesis via the Wood-Ljungdahl pathway (WLP) [1][2][3][4][5]7]. Although these studies provide valuable insights into these organisms, the presence of only a single pure culture and the scarcity of experimental studies targeting Atribacterota obscure a more comprehensive understanding of the phylum's nature. ...
... Two MAGs (HX-AS.bin.3 and HX-OS.bin.34) were assigned to the classes Atribacteria (OP9) and JS1 within the phylum Atribacterota according to the GTDB-Tk [33]. The presence of HX-AS.bin.3, a member of class Atribacteria, in the long-term acetate-amended enrichment (0.31 % in HX-AS; Table S3) is consistent with a previously proposed role of some Atribacterota in syntrophic acetate oxidation [1][2][3][4][5]7]. However, genes coding for the Wood-Ljungdahl pathway (acsABCED) were not detected in HX-OS.bin.34 and HX-AS.bin.3, which obscured their functions in these environments. ...
Background
The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood.
Results
We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments.
Conclusion
Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory.
5rnPv2dUCRDw24_qsw9GBLVideo Abstract
... Atribacteria ASVs were dominated by an unknown genus of the order JS-1 and were more common in deeper sediment fractions within or below the SMTZ. Often found in high abundance in deep subsurface biospheres, JS-1 Atribacteria have been found elevated in hydrate-bearing sediments in the region (Glass et al. 2021) and elsewhere (Ruff et al. 2015;Marlow et al. 2021), and have been shown capable of entering seep habitats through upward advection with fluid flow (Hoshino et al. 2017). A high proportion of Epsilonbacteraota ASVs within seep samples were the Campylobacterales genus Sulfurovum, a nonmat forming sulfide-oxidizing bacteria that has been found in abundance at other seeps in the region (Marlow et al. 2014;Seabrook et al. 2018). ...
In the past decade, thousands of previously unknown methane seeps have been identified on continental margins around the world. As we have come to appreciate methane seep habitats to be abundant components of marine ecosystems, we have also realized they are highly dynamic in nature. With a focus on discrete depth ranges across the Cascadia Margin, we work to further unravel the drivers of seep‐associated microbial community structure. We found highly heterogenous environments, with depth as a deterministic factor in community structure. This was associated with multiple variables that covaried with depth, including surface production, prevailing oxygen minimum zones (OMZs), and geologic and hydrographic context. Development of megafaunal seep communities appeared limited in shallow depth zones (~ 200 m). However, this effect did not extend to the structure or function of microbial communities. Siboglinid tubeworms were restricted to water depths > 1000 m, and we posit this deep distribution is driven by the prevailing OMZ limiting dispersal. Microbial community composition and distribution covaried most significantly with depth, but variables including oxygen concentration, habitat type, and organic matter, as well as iron and methane concentration, also explained the distribution of the microbial seep taxa. While members of the core seep microbiome were seen across sites, there was a high abundance of microbial taxa not previously considered within the seep microbiome as well. Our work highlights the multifaceted aspects that drive community composition beyond localized methane flux and depth, where environmental diversity adds to margin biodiversity in seep systems.
... Notably, Japan Trench sediments display a relatively shallow depth of oxygen penetration and an elevated sulfate-methane transition zone (SMTZ) due to heightened methane flux (10,13). Consequently, anaerobic heterotrophic and methanogenic metabolism plays vital roles in breaking down refractory organic matter in deeper layers (14) Atribacterota, a gram-negative bacterial phylum, has been identified as the predom inant microbial taxon in both organic-rich and anoxic sediments (15), including gas hydrates and methanogenic sediments (16)(17)(18)(19). Atribacterota includes the OP9 and JS1 classes, whose members were initially discovered in a yellowstone hot spring and deep marine sediments of the Japan Sea, respectively (20,21). ...
... Although no represen tative of JS1 class has been cultivated, single-cell amplified genomes (SAGs) of JS1 bacteria have been retrieved from various natural environments, including Sakinaw Lake, Canada (26); Etoliko Lagoon, Greece (26); Aarhus Bay, Denmark (22); and marine sediments from the Ross Sea (27). The metagenome-assembled genome (MAG) of the JS1 group in methane-hydrate-bearing sediments revealed the presence of genes encoding osmolytes, suggesting their adaptations to high pressures (18) and survival in the deep biosphere (14). Furthermore, a recent global-scale study on microbial diversity in marine sediments revealed that Atribacterota JS1 relatives are infrequently detected in aerobic sediments of open ocean gyres but are predominant in organic-rich anaerobic sediments along coastal areas (15). ...
Deep-sea and subseafloor sedimentary environments host heterotrophic microbial communities that contribute to Earth’s carbon cycling. However, the potential metabolic functions of individual microorganisms and their biogeographical distributions in hadal ocean sediments remain largely unexplored. In this study, we conducted single-cell genome sequencing on sediment samples collected from six sites (7,445–8,023 m water depth) along an approximately 500 km transect of the Japan Trench during the International Ocean Discovery Program Expedition 386. A total of 1,886 single-cell amplified genomes (SAGs) were obtained, offering comprehensive genetic insights into sedimentary microbial communities in surface sediments (<1 m depth) above the sulfate-methane transition zone along the Japan Trench. Our genome data set included 269 SAGs from Atribacterota JS1, the predominant bacterial clade in these hadal environments. Phylogenetic analysis classified SAGs into nine distinct phylotypes, whereas metagenome-assembled genomes were categorized into only two phylotypes, advancing JS1 diversity coverage through a single cell-based approach. Comparative genomic analysis of JS1 lineages from different habitats revealed frequent detection of genes related to organic carbon utilization, such as extracellular enzymes like clostripain and α-amylase, and ABC transporters of oligopeptide from Japan Trench members. Furthermore, specific JS1 phylotypes exhibited a strong correlation with in situ methane concentrations and contained genes involved in glycine betaine metabolism. These findings suggest that the phylogenomically diverse and novel Atribacterota JS1 is widely distributed in Japan Trench sediment, playing crucial roles in carbon cycling within the hadal sedimentary biosphere.
IMPORTANCE
The Japan Trench represents tectonically active hadal environments associated with Pacific plate subduction beneath the northeastern Japan arc. This study, for the first time, documented a large-scale single-cell and metagenomic survey along an approximately 500 km transect of the Japan Trench, obtaining high-quality genomic information on hadal sedimentary microbial communities. Single-cell genomics revealed the predominance of diverse JS1 lineages not recoverable through conventional metagenomic binning. Their metabolic potential includes genes related to the degradation of organic matter, which contributes to methanogenesis in the deeper layers. Our findings enhance understanding of sedimentary microbial communities at water depths exceeding 7,000 m and provide new insights into the ecological role of biogeochemical carbon cycling in the hadal sedimentary biosphere.
... These processes are (HM1, HM3, HM5, HM_SQ, S11, SY5, and SY6) and site F cold seep (RS, SF, FR, and SF_SQ). Paired-end sequencing data from ENP, SMM, WGM, NGM, HM, MS, LS and part of site F (RS and FR) were downloaded from the National Center for Biotechnology Information-Sequence Read Archive (NCBI-SRA) and European Bioinformatics Institute-European Nucleotide Archive (EBI-ENA) according to the accession numbers published in each study [8][9][10][22][23][24][25][26] . The remaining 106 metagenomic datasets used in this study were obtained from our previous publications 7,14,[27][28][29][30][31][32][33][34] . ...
Cold seeps harbor abundant and diverse microbes with tremendous potential for biological applications and that have a significant influence on biogeochemical cycles. Although recent metagenomic studies have expanded our understanding of the community and function of seep microorganisms, knowledge of the diversity and genetic repertoire of global seep microbes is lacking. Here, we collected a compilation of 165 metagenomic datasets from 16 cold seep sites across the globe to construct a comprehensive gene and genome catalog. The non-redundant gene catalog comprised 147 million genes, and 36% of them could not be assigned to a function with the currently available databases. A total of 3,164 species-level representative metagenome-assembled genomes (MAGs) were obtained, most of which (94%) belonged to novel species. Of them, 81 ANME species were identified that cover all subclades except ANME-2d, and 23 syntrophic SRB species spanned the Seep-SRB1a, Seep-SRB1g, and Seep-SRB2 clades. The non-redundant gene and MAG catalog is a valuable resource that will aid in deepening our understanding of the functions of cold seep microbiomes.
... Additionally, we observed the methanotroph g__Kmv04 (Methanocomedenaceae). We also found members of the bacterial phyla Atribacterota (g__CG2-30-33-13) and Aerophobota (g__AE-B3A), which are commonly found in methane-rich sediments across various environments such as temperate soils, deep marine sediments, and permafrost 39,40 . We detected several methanotrophic bacteria with high detection, including high-affinity methanotrophs like Methyloceanibacter [41][42][43] and genera from Methylomonadaceae such as g__KS41, which are commonly found in acidic forest soils 44 , and Methyloglobulus, found in lake sediments 45 . ...
Using ancient environmental DNA (eDNA) we reconstructed microbial and viral communities from the Kap København Formation in North Greenland. We find pioneer microbial communities, along with likely dormant methanogens from the permafrost's seed bank. Our findings reveal that at the time of the formation, the terrestrial input of the Kap København site originated from a palustrine wetland, suggesting non-permafrost conditions. During this time, detection of methanogenic archaea and carbon processing pathways suggests a moderate strengthening of methane emissions through the northward expansion of wetlands. Intriguingly, we discover a remarkable sequence similarity (>98%) between pioneer methanogens and present-day thawing permafrost counterparts. This suggests that not all microbes respond uniformly to environmental change over geological timescales, but that some microbial taxa's adaptability and resilience remain constant over time. Our findings further suggest that the composition of microbial communities is changing prior to plant communities as a result of global warming.
... Using porewater geochemistry profiles, the SMTZ is generally defined by the overlapping zone between dissolved sulfate and methane, and its depth where both concentrations are equimolar (Egger et al., 2018;Jørgensen et al., 2019). In practice, however, different studies use different ranges (e.g., Hong et al., 2013;Yoshinaga et al., 2014) and/or units (e.g., Glass et al., 2021;Johnson et al., 2021) to plot methane and sulfate concentration, and then assign the SMTZ depth as the intersection of two profiles. This ad hoc definition can lead to a SMTZ depth that is shallower than the depth indicated by equimolar concentration (see Jørgensen et al., 2019). ...
... The depth of SMTZ (solid horizontal black line) is defined as the intercept between sulfate and methane regressions fits, with the uncertainty (gray band) calculated as the overlapping area of both 95% confidence intervals. For comparison, previously reported SMTZ depths (colored line) and intervals (colored band) from the same site (Chatterjee et al., 2011;Glass et al., 2021;Tréhu et al., 2003) are shown to the right. (Table 2). ...
... Gas hydrates, ice-like crystalline solids composed of water and hydrocarbons, are widely discovered in the deep subseafloor of every continental margin [1,2], typically hundreds of meters below the seafloor, e.g., the Hydrate Ridge [3] and Nankai Trough [4,5]. Ecological studies based on single marker genes have revealed abundant and diversified members of archaea and bacteria in the deep subsurface sediments associated with gas hydrates over the global ocean [3][4][5][6][7]. ...
... Gas hydrates, ice-like crystalline solids composed of water and hydrocarbons, are widely discovered in the deep subseafloor of every continental margin [1,2], typically hundreds of meters below the seafloor, e.g., the Hydrate Ridge [3] and Nankai Trough [4,5]. Ecological studies based on single marker genes have revealed abundant and diversified members of archaea and bacteria in the deep subsurface sediments associated with gas hydrates over the global ocean [3][4][5][6][7]. All known forms of life require sources of carbon and energy to thrive. ...
... The transition from Chloroflexota to Caldatribacteriota between redox zones along with sediment depth implies intense subseafloor selection [49]. Members of Caldatribacteriota are likely more adaptive to deep anoxic sediments with less organic carbon and energy availability [3,50], which explains their successful survival in the deep subseafloor. ...
Background
Gas hydrate-bearing subseafloor sediments harbor a large number of microorganisms. Within these sediments, organic matter and upward-migrating methane are important carbon and energy sources fueling a light-independent biosphere. However, the type of metabolism that dominates the deep subseafloor of the gas hydrate zone is poorly constrained. Here we studied the microbial communities in gas hydrate-rich sediments up to 49 m below the seafloor recovered by drilling in the South China Sea. We focused on distinct geochemical conditions and performed metagenomic and metatranscriptomic analyses to characterize microbial communities and their role in carbon mineralization.
Results
Comparative microbial community analysis revealed that samples above and in sulfate-methane interface (SMI) zones were clearly distinguished from those below the SMI. Chloroflexota were most abundant above the SMI, whereas Caldatribacteriota dominated below the SMI. Verrucomicrobiota, Bathyarchaeia, and Hadarchaeota were similarly present in both types of sediment. The genomic inventory and transcriptional activity suggest an important role in the fermentation of macromolecules. In contrast, sulfate reducers and methanogens that catalyze the consumption or production of commonly observed chemical compounds in sediments are rare. Methanotrophs and alkanotrophs that anaerobically grow on alkanes were also identified to be at low abundances. The ANME-1 group actively thrived in or slightly below the current SMI. Members from Heimdallarchaeia were found to encode the potential for anaerobic oxidation of short-chain hydrocarbons.
Conclusions
These findings indicate that the fermentation of macromolecules is the predominant energy source for microorganisms in deep subseafloor sediments that are experiencing upward methane fluxes.
84_GYpCEs37AndHmKLQLsZVideo Abstract
... The microbial community is drastically different below the sulfate-methane transition zones than in shallow sediments. In deep subsurface hydrate sediments, Atribacterota (JS-1) and Chloroflexota dominate bacterial 16S rRNA gene sequences and Asgardarchaeota (formerly MBGB/DSAG) dominate archaeal 16S rRNA gene sequences; these trends have been observed for deep subsurface hydrate-bearing sediments around the Pacific Rim, including Nankai Trough (Katayama et al., 2016;Wellsbury et al., 2000), Umitaka Spur (Yanagawa et al., 2014), Ulleung Basin (Lee et al., 2013), Cascadia Margin (Parkes et al., 2014), Hydrate Ridge (Glass et al., 2021;Inagaki et al., 2006;Nunoura et al., 2008), and the Peru Margin (Inagaki et al., 2006). 16S rRNA gene sequences from the South China Sea differed between studies (Cui et al., 2019(Cui et al., , 2020Gong et al., 2017;Jiang et al., 2007;Jiao et al., 2015), but metagenomic sequencing shows dominance of Atribacterota (JS-1), Chloroflexota, and Asgardarchaeota (Zhang et al., 2022), consistent with other Pacific Rim sites. ...
... Microbial metabolisms and stress adaptions in hydrate sediments have been assessed with metagenomics, metagenome-assembled geno mes, and metatranscriptomics, revealing that most microbes that reside in hydrate-bearing sediments are heterotrophs that ferment macromolecules (Dong et al., 2019;Zhang et al., 2022). However, a respiratory complex predicted to couple proton and sodium translocation to molecular hydrogen production using an evolutionarily distinct hydrogenase has been found in JS-1 class of Atribacterota and several Firmicutes under Hydrate Ridge (Glass et al., 2021). For survival in salt stress caused by salt exclusion during hydrate formation (Ussler & Paull, 2001), microbial production and transport of the osmolyte trehalose appears to be common in methane-rich marine sediments (Bird et al., 2019;Chen et al., 2019;Glass et al., 2021). ...
... However, a respiratory complex predicted to couple proton and sodium translocation to molecular hydrogen production using an evolutionarily distinct hydrogenase has been found in JS-1 class of Atribacterota and several Firmicutes under Hydrate Ridge (Glass et al., 2021). For survival in salt stress caused by salt exclusion during hydrate formation (Ussler & Paull, 2001), microbial production and transport of the osmolyte trehalose appears to be common in methane-rich marine sediments (Bird et al., 2019;Chen et al., 2019;Glass et al., 2021). ...
Methane hydrates host unique microbial ecosystems in the deep subsurface on Earth and potentially on other planetary bodies. The source of the methane in these hydrates is largely microbial, yet methanogenic archaea comprise a tiny fraction of the microbial community. Bacteria - namely members of the JS-1 class of Atribacterota - have repeatedly been found to dominate the microbial community in deep hydrate sediments from continental margins. Microbes in hydrate sediments harbor adaptations to survive salt stress, such as synthesis and transport of osmolytes. Burning questions remain about this highly flammable subject, particularly regarding the role of microbes in the formation and stability of methane hydrates. This article is protected by copyright. All rights reserved.
... Following upward transport of Caldatribacteriota, their higher sequence abundance compared to other bacteria in shallow sediments (Figure 3) may additionally be due to a competitive advantage from being adapted to the stronger selective pressures of the deep subsurface. Caldatribacteriota have unique attributes that may provide advantages in the energylimited deep subsurface (Bird et al. 2019) as well as a variety of other environmental stresses (Glass et al. 2021), including hypothesized behaviour of carbohydrate storage as so-called 'selfish bacteriaʼ (Orsi 2018). ...
Deep sea hydrocarbon seep detection relies predominantly on geochemical analyses of seabed marine sediment cores to identify the presence of gas or oil. The presence of seeping hydrocarbons in these locations alters resident microbial community structure, leading to culture-based biodegradation assays as a complement to geochemical tools for seep detection. Biodiversity surveys of microbial communities can offer a similar proxy for seeping hydrocarbons, but this strategy has not been extensively investigated in deep water settings. In this study, 16S rRNA gene sequencing of bacterial communities was performed on sediment cores obtained in >2500 m water depth at 43 different locations in the NW Atlantic Ocean. Core samples from as deep as 10 metres below seafloor (mbsf) were assessed for gas composition, gas isotopes and liquid hydrocarbons. Over 650 bacterial 16S rRNA gene amplicon libraries were constructed from different sediment depths at these locations. Select sites showed strong evidence for the presence of thermogenic or biogenic hydrocarbons such that bacterial population analyses revealed significant differences between hydrocarbon seep and non-seep locations. Specific bacterial indicators were associated with different sediment depth intervals. Caldatribacteriota and Campilobacterota OTUs were observed in high relative sequence abundance in hydrocarbon seep sediments, particularly in the 20-50 cmbsf interval. Furthermore, these groups were differentially abundant between sites with thermogenic and biogenic hydrocarbons. The patterns revealed here suggest that microbial screening has the potential to play a key role in hydrocarbon seep detection and characterisation in remote deep-sea environments.
... While members of the Caldatribacteriota phylum are prevalent in cold seep sediments 11,40 , no previous studies have inferred that they are diazotrophic. These results highlight that Caldatribacteriota may play biogeochemically and ecologically significant roles within diverse cold seeps besides their role in carbon cycling 66 . Most other diazotrophs are at lower abundance (<1% of the microbial community). ...
... Metagenomes were compiled from 61 deep-sea sediment samples (water depths ranging from 860-3005 m) collected from 11 geographically diverse cold seep sites from around the world (Fig. 1, Supplementary Data 1, and references therein). Part of these metagenomes were downloaded from NCBI's Sequence Read Archive, including datasets derived from Haakon Mosby mud volcano, Eastern North Pacific ODP site 1244, Mediterranean Sea Amon mud volcano, Santa Monica Mounds, and Gulf of Mexico 66,[82][83][84][85] . Other data were obtained from our previous publications described in detail ...
Microbially mediated nitrogen cycling in carbon-dominated cold seep environments remains poorly understood. So far anaerobic methanotrophic archaea (ANME-2) and their sulfate-reducing bacterial partners (SEEP-SRB1 clade) have been identified as diazotrophs in deep sea cold seep sediments. However, it is unclear whether other microbial groups can perform nitrogen fixation in such ecosystems. To fill this gap, we analyzed 61 metagenomes, 1428 metagenome-assembled genomes, and six metatranscriptomes derived from 11 globally distributed cold seeps. These sediments contain phylogenetically diverse nitrogenase genes corresponding to an expanded diversity of diazotrophic lineages. Diverse catabolic pathways were predicted to provide ATP for nitrogen fixation, suggesting diazotrophy in cold seeps is not necessarily associated with sulfate-dependent anaerobic oxidation of methane. Nitrogen fixation genes among various diazotrophic groups in cold seeps were inferred to be genetically mobile and subject to purifying selection. Our findings extend the capacity for diazotrophy to five candidate phyla (Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262), and suggest that cold seep diazotrophs might contribute substantially to the global nitrogen balance. Microbial nitrogen fixation could be important in the deep sea. Here the authors investigate metagenomes and metatranscriptomes of diazotrophs from deep sea cold seep sediments, reveal greater phylogenetic and functional diversity than hitherto known.
