Biology subjects

Orphan, V. J.

Publications and source records attributed to Orphan, V. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Microcalorimetry reveals bacterial motility as an expensive trait during starvation

Quantifying the energetic costs of cellular traits is important to understand their evolutionary constraints and ecological function. However, direct measurements of energy expenditure of distinct traits are rare. Here, we present direct measurements of the power dissipation due to bacterial flagellar motility using microcalorimetry, based on measuring the power dissipation of a motile population and a non-motile mutant. For the single-flagellated marine bacterium \textit{Vibrio coralliilyticus}, we determined the power dissipated by motility in cultures to be 0.91 W/mL/OD. Per swimming cell this amounts to 2.0E-15 W, or a turnover of 2.5E4 ATP molecules per second. The power dissipated for motility equals 35 % of total metabolism during starvation, indicating this is likely the most expensive cellular trait during starvation. The dissipation for motility is in good agreement with that predicted using resistive force theory, indicating that any thermodynamic inefficiencies upstream of the flagellar motor, such as metabolic inefficiencies, are small. We further show that the multiflagellated bacterium Escherichia coli dissipates 0.70 W/mL/OD when in motility buffer, comparable to marine bacteria despite the much lower swimming speed, and hence indicating lower efficiency of swimming. Finally, we show that the energy spent by Vibrio coralliilyticus on motility can have major consequences on their survival under starvation, causing a 40 % reduction in survival after 3 weeks of starvation of a motile compared to non-motile mutant. These results reveal the large costs involved in an individual trait that shapes microbial life in heterogeneous environments.

biophysics↗

bioRxiv2025

Cycling of sulfur redox intermediates drives microbial activity in the sulfate-methane transition zone of cold methane seeps

Microbial sulfate reduction is a cornerstone of marine sediment biogeochemistry, driving carbon remineralization and fueling the anaerobic oxidation of methane (AOM). Yet in zones of high methane flux, sulfate limitations may constrain the sulfate-reducing bacteria (SRB) and anaerobic methanotrophic archaea (ANME) that typically perform AOM. Although often overlooked, sulfur redox intermediates are readily utilized by diverse microorganisms, potentially driving AOM in sulfate-limited zones. To resolve the microbial mechanisms underlying cryptic sulfur cycling in such sediments, Monterey Canyon cold methane seeps were investigated through an integrated geochemical, isotopic, and metatranscriptomic approach. High-resolution electrochemical measurements confirmed intense sulfide production in seep sites, and long-term anoxic incubations were conducted with sediment from the SMTZ amended with elemental sulfur, thiosulfate, or sulfate as the sole sulfur source, with or without methane. Over 650 days, sulfide accumulation was greatest in elemental sulfur treatments, followed by thiosulfate and sulfate; in all cases methane addition enhanced sulfide production. Isotopic measurements showed modest S-isotope fractionation indicating that the large fractionations typical of slow sulfate reduction were muted by additional sulfur transformations. In elemental sulfur treatments, isotopic and geochemical patterns suggested that disproportionation was unlikely. Metatranscriptomes revealed broad expression of sox genes and abundant dsr/apr across treatments, along with thiosulfate-linked phs upregulation. While ANME-2c and SEEP-SRB2 activity increased with methane, transcriptomic and isotopic data together highlighted the roles of Desulfocapsaceae, Desulfobulbaceae, and Sulfurovaceae lineages in mediating sulfur transformations. Taken together, these results demonstrate how cryptic sulfur cycling may sustain microbial communities in sulfate-depleted deep-sea sediments and contribute to AOM.

ecology↗

bioRxiv2025

Carbon monoxide oxidation expands the known metabolic capacity in anaerobic methanotrophic consortia

Efficient energy metabolism is essential for microorganisms living on the thermodynamic edge. Consortia of anaerobic methane-oxidizing archaea (ANME-2) and sulphate-reducing bacteria (SRB) represent globally relevant syntrophic associations capable of growing with minimal amounts of free energy and can persist when methane becomes limiting. Their potential for physiological plasticity, including the use of electron donors beyond methane is poorly understood. Carbon monoxide (CO) has been reported in seep environments and represents a thermodynamically favourable alternative electron donor due to its low reduction potential. We demonstrate that environmental ANME-SRB consortia can oxidize CO in the absence of methane, in anoxic microcosm experiments using a combination of stable isotope geochemical tracers, metatranscriptomics, and single cell activity measurements (FISH-nanoSIMS). The oxidation of CO was coupled with sulphate-reduction by syntrophic consortia, and, in the absence of sulphate, through CO2 reduction to methane by ANME-2. Under these conditions, the production of methane was one ninth the rate of methanotrophy coupled to sulphate-reduction. Paired single cell FISH-nanoSIMS analysis of anabolic activity indicates that CO respiration appears to support cell maintenance rather than active growth, consistent with the observed down-regulation of energy generating complexes in ANME (e.g., mtr, rnf, etc.). The versatile capability of CO oxidation by anaerobic methanotrophic consortia broadens our understanding of carbon cycling in methane seeps and highlights potential mechanisms of resilience by methanotrophic archaea under changing geochemical regimes.

microbiology↗

bioRxiv2025

Phage-mediated lysis increases growth rate of surviving bacterial cells

Bacterial phage infection and subsequent lysis are traditionally considered mechanisms of bacterial mortality and viral propagation; additionally, emerging evidence indicates that they may also contribute to nutrient recycling in broader ecological systems. However, it remains unclear how the nutrients released during cell lysis affect the growth dynamics of the remaining bacterial population. Addressing this gap, we built a controlled system consisting of two Escherichia coli lysogenic strains: one carrying a wild-type{lambda} prophage and the other a temperature-inducible variant that can be induced to lyse at 38 {degrees}C. Using this system, we selectively induced phage lysis in a defined fraction of the population and quantified both total biomass and the biomass of surviving, non-lysed cells. We observed that the biomass loss was consistently smaller than expected based on the fraction of lysed cells, supporting the idea that some of the released biomass is rapidly recycled by the non-lysed population. To formalize the observed dynamics and obtain quantitative insight, we developed a mathematical model showing that nutrients released during lysis can transiently enhance the growth rate of the surviving, non-lysed cells. This effect emerges on a short timescale of minutes, consistent with the rapid onset of biomass compensation observed experimentally. The growth rate increase was confirmed in single-cell experiments using microfluidics and time-lapse microscopy, where we cultured wild-type lysogens in lysate-containing culture supernatants. In summary, the results suggest that nutrients released through lysis are rapidly consumed, leading to an acceleration in the growth rate of non-lysed cells. The consequent partial compensation for cell loss can substantially influence the population dynamics, highlighting phage lysis as a direct modulator of bacterial growth. Overall, our findings provide quantitative insights into how phage-mediated lysis affects the physiology of non-lysed bacterial cells, extending beyond its well-established role in biomass recycling.

microbiology↗

bioRxiv2025

Illuminating the active virosphere with BONCAT and single virus genomic sequencing technologies

Marine viruses impact biogeochemical cycles through cell lysis, releasing organic matter and nutrients that fuel ocean productivity. Identifying and quantifying the specific viruses active in these processes remains a priority in the field. Here, we introduce a click-chemistry method to fluorescently label, sort, and sequence the genomes of newly produced viral particles released from transcriptionally active host microbial cells, alongside the analysis of co-occurring inactive cells and viruses in environmental samples. This approach, called viral BONCAT-FACS, combines biorthogonal non-canonical amino acid tagging (BONCAT) with environmental sample incubation, followed by single-virus and single-cell sorting by flow cytometry (FACS). Genomic analysis of translationally-active cells and new viral progeny in coastal seawater incubations confirmed BONCAT labeling and successful sorting of diverse marine bacteria, microeukaryotic cells, and virioplankton, with stark differences in the predicted turnover of specific groups of infecting viruses, including Pelagiphages, Methylophages, a Flavobacteriales-associated novel "Far-T4" clade, algae-infecting giant NCLDV viruses, and parasitic virophages. Sequenced BONCAT-active cells showed a strong enrichment in viral contigs relative to the inactive cell fraction, suggestive of a large proportion of translationally-active virocells. This study illustrates the effectiveness of viral BONCAT-FACS for uncovering genome-resolved viral-host dynamics. By providing a direct approach for tracking active viral infections in natural environments, this method enhances our ability to investigate behavior and interactions of these nanoscale predators, expanding our understanding of their role in ecosystem dynamics.

microbiology↗

bioRxiv2025

Viral communities from long-term anaerobic alkane-oxidizing enrichments may promote cell surface adhesion

The anaerobic oxidation of methane and higher C2+ alkanes is a dominant metabolism within hydrocarbon-rich deep-sea sediments and is largely mediated by alkane-oxidizing archaea in metabolic partnership with syntrophic sulfate-reducing bacteria. While these processes feed a diverse ecosystem, the viral component of alkane-rich sediments has historically been overlooked. We analyzed the viral community in long-term sediment-free enrichments of alkane-degrading organisms and found that abiotic factors such as incubation temperature had a greater correlation with community composition than with the phylogenetic patterns among individual viral species. No auxiliary metabolic genes directly involved in hydrocarbon oxidation or sulfate reduction were found, but the presence of AMGs involved in heme synthesis pathways common in methane oxidizers hints at a possible viral impact on alkane degradation. We also found evidence supporting the presence of a viral genus infecting several phyla across both the bacterial and archaeal domains, including one of the sulfate-reducing bacterial partners in the alkane-oxidizing syntrophy. Lastly, we report the presence of nosD-like proteins in viruses from sediment-derived systems that are not present in water column datasets; their distribution, genomic context, and lack of canonical nosD characteristics suggest an alternate adhesion-related role in sediment communities. The number of novel viruses obtained from these enrichment cultures and their potential roles in mediating host physiology illustrate the importance of studying the viral component in laboratory and environmental systems.

ecology↗

bioRxiv2025

Deep sea anaerobic microbial community couples the degradation of insoluble chitin to extracellular electron transfer

Chitin is a major structural component of arthropod exoskeletons, and an important carbon and nitrogen source in marine environments. In anoxic sediments, its degradation generates chitooligosaccharides and N-acetylglucosamine (GlcNAc), which are fermented into smaller organic molecules and oxidized anaerobically using soluble electron acceptors or insoluble ones such as metal oxides. To date, many aspects of chitin degradation in deep-sea anoxic sediments have been overlooked, including the potential coupling of insoluble chitin degradation to metal oxide reduction, the involvement of extracellular electron transfer (EET), and the spatial organization of the microorganisms involved. Using anoxic deep-sea sediments recovered from a whale fall site, we developed an innovative workflow based on electrochemical reactors, to characterize chitin degradation in these environments. Sediment samples enriched on poorly crystalline iron oxides, and subsequently transferred into an electrochemical reactor poised at +0.22 V vs SHE, showed active anodic current production when supplied with chitin, which increased 2-fold when amended with GlcNAc. Chitin reactors were dominated by Vallitalea (Firmicutes), Spirochaetota, Gammaproteobacteria and Desulfobacterota. Exoenzyme activity assays, metabolite profiling, and continued anodic current production confirmed ongoing chitin degradation linked to EET. We observed metabolic associations between chitin degraders and secondary consumers using in situ imaging (16S rRNA gene FISH coupled with BONCAT and nanoSIMS). These microbial partners, within the electrode-attached community, required close proximity to the poised electrode ([≤] 10 {micro}m) to remain metabolically active. Supporting these observations, cultured isolates of Vallitalea sp. and Trichloromonas sp. recovered from the whale fall site exhibited chitin degradation and electrochemical activity, respectively. When co-cultured in an bioelectrochemical reactor, the acetate produced by Vallitalea sp. during chitin degradation fueled Trichloromonas sp., which facilitated EET, hereby demonstrating that syntrophic interactions are used to couple anoxic chitin degradation to EET in deep-sea sediments. These findings exemplify the interspecies interactions and resource optimization occurring in hard-to-reach and largely unknown deep-sea ecosystems.

microbiology↗

bioRxiv2025

The Ambiguous Genetic Code of Methanogenic Archaea that Grow on Methylamines

Natural genetic code expansion is a phenomenon wherein an additional amino acid is encoded by a stop codon. These non-standard amino acids are beneficial as they facilitate novel biochemical reactions. However, code expansion leads to ambiguity at the recoded stop codon, which can either be read through or terminated. Pyrrolysine (Pyl) is encoded by the amber codon (TAG/UAG) and is widespread in archaea, where it is required for methylamine-mediated methanogenesis, an environmentally important metabolism. Mechanisms to conditionally suppress the amber stop codon for Pyl installation during protein synthesis have not been identified. Using the model methanogen, Methanosarcina acetivorans, we demonstrate that Pyl-encoding archaea maintain an ambiguous genetic code wherein UAG encodes dual meaning as stop and Pyl. Our data suggest that expression of Pyl biosynthesis and incorporation genes is tuned to the cellular demand for Pyl, which allows these archaea to navigate ambiguous stop decoding in response to environmental cues.

microbiology↗

bioRxiv2025

Diversity and single-cell activity of endolithic microbes in sediment-hosted carbonate nodules within and below the sulfate-methane transition zone

Authigenic carbonate concretions ( nodules) precipitate in marine seep sediments as a result of anaerobic oxidation of methane (AOM). These rocks host active endolithic microbial communities and persist as important methane sinks. Still, how these communities and their activity differ from those in adjacent seep sediments, particularly as a function of proximity to the sulfate-methane transition zone (SMTZ), remains understudied. Here, we sampled sediments and nodules within and below the SMTZ (0-57 cm deep) at four active deep-sea seep areas in Santa Monica Basin, CA. Measurements of high nodule porosities (43-51%) coupled to strong similarities between sediment and nodule 16S rRNA-based community profiles, including below the SMTZ, suggest continued perfusion and exchange between buried nodules and surrounding sediment. Shared, depth-dependent transitions in methanotrophic taxa (ANME-1, ANME-2, ANME-3) and methanogenic taxa (Methanofastidiosales) below the SMTZ were also consistent with trends in porewater methane and sulfate concentrations, porewater DIC, and nodule {delta}13C values -- underscoring the impact of different geochemical conditions on community structure and suggestive of under-characterized physiological plasticity in ANME-1. Laboratory-based BONCAT incubations of nodules within the SMTZ over [~]14-weeks revealed active sulfide production and translationally active endolithic microorganisms. However, cells from parallel nodule incubations recovered below the SMTZ showed weak-to-negligible BONCAT-based activity despite similar cell abundances -- suggestive of low activity on shorter timescales or dormancy. Together, these data challenge the interpretation of passively recorded microbiological signatures in seep sediment-hosted carbonate nodules and expand our understanding of how these endolithic communities may be actively shaped by past and present conditions. ImportanceThis study advances earlier investigations of microbial communities in buried seep carbonate nodules by integrating microbiological profiles of nodules and sediments, sediment geochemistry, single-cell activity measurements, and nodule mineral, geochemical, and physical characteristics within and below the sulfate-methane transition zone in deep-sea methane seeps. This approach allows us to view how nodule-hosted, endolithic microbial communities change relative to their surrounding sediments across multiple geochemical contexts and better understand how formation history and environmental conditions might affect community identity and metabolic function. Results indicate that the seep nodule and surrounding sediment communities are closely linked across diverse geochemical conditions. This connectivity between sediments and carbonate nodules is distinct from that observed in exhumed seep carbonates, with implications regarding how microbial community composition within these nodules are interpreted, suggesting that instead of a passive recorder of the communities at the time of formation, these nodules appear to retain diverse, metabolically viable communities.

microbiology↗

bioRxiv2025

Distinct Microbial Communities Within and On Seep Carbonates Support Long-term Anaerobic Oxidation of Methane and Novel pMMO Diversity

At methane seeps worldwide, syntrophic anaerobic methane-oxidizing archaea and sulfate-reducing bacteria (ANME-SRB) promote carbonate precipitation and rock formation, acting as methane and carbon sink. While maintenance of active anaerobic oxidation of methane (AOM) within seep carbonates has been documented, the ANME-SRB reactivity to methane exposure remains uncertain. Surface-associated microbes may metabolize AOM-derived sulfide, maintain carbonate anoxia, and contribute to carbonate dissolution and higher trophic levels; however, these microbial communities are poorly described thus far. Here we provide new insights into microbial diversity, metabolic potential, activity, and resiliency within and on Southern Californian methane seep carbonates, by combining 16S rRNA and metagenomic sequencing, laboratory incubations, and BONCAT-FISH. Ca. Methanophaga (ANME-1) dominated the carbonate interiors across different seepage activities, based on sequencing, while the dominant SRB was Ca. Desulfaltia, potentially a new ANME partner. BONCAT-FISH revealed differences in ANME-1 cell activity, suggesting cell dormancy or DNA preservation at less active seep sites. Carbonate incubations from low activity seeps ([≥]24 months) showed an exponential AOM reactivation (44-day doubling time), suggesting seep carbonates remain potential methane sinks over dynamic seepage conditions. The surface-associated communities were distinct from the carbonate interior and other seep habitats, and highly heterogeneous. Surface ANME-SRB biofilms and sulfide-oxidizing bacterial mats were associated with high and intermediate AOM carbonates, potentially influencing carbonate precipitation/dissolution. Carbonate surfaces shared diverse aerobic methanotrophs with invertebrates, potentially serving as pool for animal epibionts. Besides particulate methane monooxygenases from aerobic methanotrophs, we found divergent forms including within a Methylophagaceae (GCA-002733105) MAG suggesting a new function within Methylophagaceae.

ecology↗

bioRxiv2024

CABO-16S : A Combined Archaea, Bacteria, Organelle 16S database for amplicon analysis of prokaryotes and eukaryotes in environmental samples

Identification of both prokaryotic and eukaryotic microorganisms in environmental samples is currently challenged by either the burden of additional sequencing required to obtain both 16S and 18S rRNA sequences or the introduction of multiple biases induced by the use of "universal" primers. Organellar 16S rRNA sequences are automatically amplified and sequenced along with prokaryote 16S rRNA, and may provide an alternative method to identify eukaryotic microorganisms. CABO-16S combines bacterial and archaeal sequences from the SILVA database with 16S rRNA sequences of plastids and other organelles from the PR2 database to enable identification of all 16S rRNA sequences. Comparison of CABO-16S with SILVA 138.2 results in equivalent taxonomic classification of mock communities and increased classification of diverse environmental samples. In particular, identification of phototrophic eukaryotes in shallow seagrass environments, marine waters, and lake waters was increased. CABO-16S also provides the framework to add curated datasets of specialized sequences for further classification of clades which are not currently included in other databases. Addition of sequences obtained from Sanger sequencing of methane seep sediments and curated sequences of the polyphyletic SEEP-SRB1 clade resulted in differentiation of syntrophic and non-syntrophic SEEP-SRB1 in hydrothermal vent sediments. Such additions may simplify analysis of communities contributing to the anaerobic oxidation of methane, and highlight the potential benefit of amending existing training sets with curated sequences when studying extreme or unique environments underrepresented in existing databases.

microbiology↗

bioRxiv2024

Spatial evidence of cryptic methane cycling and methylotrophic metabolisms along a land-ocean transect in a California coastal wetland

Methylotrophic methanogenesis in the sulfate reduction zone of coastal and marine sediments couples with anaerobic methane oxidation (AOM), forming the cryptic methane cycle. This study provides evidence of cryptic methane cycling in the sulfate-reducing zone across a land-ocean transect of four stations--two brackish, one marine, and one hypersaline--within the Carpinteria Salt Marsh Reserve (CSMR), Southern California, USA. The top 20 cm of sediment from the transect underwent geochemical and molecular (16S rRNA) analyses, in-vitro methanogenesis incubations, and radiotracer incubations using 35S-SO4, 14C-mono-methylamine, and 14C-CH4. Sediment methane concentrations were consistently low (3 to 28 {micro}M) except at the marine station, where they increased with depth (max 665 {micro}M). Methanogenesis from mono-methylamine was detected throughout the sediment at all stations with estimated rates ranging between 0.14 and 3.8 nmol cm-3 d-1. 16S rRNA analysis identified methanogenic archaea capable of producing methane from methylamines in sediment where methanogenesis was found to be active. Metabolomic analysis of porewater showed mono-methylamine was mostly undetectable (<3 {micro}M) or present in trace amounts (<10 {micro}M) suggesting rapid metabolic turnover. In-vitro methanogenesis incubations showed no linear methane buildup, suggesting a process limiting methane emissions. AOM activity, measured with 14C-CH4, overlapped with methanogenesis from mono-methylamine activity at all stations, with rates ranging from 0.03 to 19.4 nmol cm-3 d-1. Porewater geochemical analysis showed the CSMR sediments are rich in sulfate and iron. Porewater sulfate concentrations (9-91 mM) were non-limiting across the transect, which support members of sulfate-reducing bacteria and likely responsible for sulfate reduction activity (1.5-2,506 nmol cm-3 d-1) at all stations in the CSMR. Porewater sulfide and iron (II) profiles indicated that the sediment transitioned from a predominantly iron-reducing environment at the two brackish stations to a predominantly sulfate-reducing environment at the marine and hypersaline station. AOM activity was likely coupled to sulfate and possibly iron reduction, coinciding with the presence of anaerobic methanotrophs and bacteria involved in these reductions. 16S rRNA analysis identified anaerobic methanotrophs at the marine and hypersaline stations, where they coexisted with putative methanogens, suggesting both groups, or methanogens alone, may be involved in cryptic methane cycling, preventing significant methane buildup in the sulfate-reducing zone. Differences in rate constants from 14C radiotracer incubations suggest a non-methanogenic process oxidizing mono-methylamine to inorganic carbon, likely mediated by sulfate-reducing bacteria. Understanding the potential competition of sulfate reducers with methanogens for mono-methylamine needs further investigation as it might be another important process responsible for low methane emissions in salt marshes.

microbiology↗

bioRxiv2024

Convergence and horizontal gene transfer drive the evolution of anaerobic methanotrophy in archaea

1Despite their large environmental impact and multiple independent emergences, the processes leading to the evolution of anaerobic methanotrophic archaea (ANME) remain unclear. This work uses comparative metagenomics of a recently evolved but understudied ANME group, Candidatus Methanovorans (ANME-3) to identify evolutionary processes and innovations at work in ANME which may be obscured in earlier evolved lineages. Within members of Methanovorans, we identified convergent evolution in carbon and energy metabolic genes as likely drivers of ANME evolution. We also identified erosion of genes required for methylotrophic methanogenesis along with horizontal acquisition of multi-heme cytochromes and other loci uniquely associated with ANME. The assembly and comparative analysis of multiple Methanovorans genomes offers important functional context for understanding the niche-defining metabolic differences between methane-oxidizing ANME and their methanogen relatives. Furthermore, this work illustrates the multiple evolutionary modes at play in the transition to a novel and globally important metabolic niche.

microbiology↗

bioRxiv2022

Physiological adaptation of sulfate reducing bacteria in syntrophic partnership with anaerobic methanotrophic archaea

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we explore the metabolic adaptation of four syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a and Seep-SRB1g) from a phylogenomics perspective, tracing the evolution of conserved proteins in the syntrophic SRB clades, and comparing the genomes of syntrophic SRB to their nearest evolutionary neighbors in the phylum Desulfobacterota. We note several examples of gain, loss or biochemical adaptation of proteins within pathways involved in extracellular electron transfer, electron transport chain, nutrient sharing, biofilm formation and cell adhesion. We demonstrate that the metabolic adaptations in each of these syntrophic clades are unique, suggesting that they have independently evolved, converging to a syntrophic partnership with ANME. Within the clades we also investigated the specialization of different syntrophic SRB species to partnerships with different ANME clades, using metagenomic sequences obtained from ANME and SRB partners in individual consortia after fluorescent-sorting of cell aggregates from anaerobic sediments. In one instance of metabolic adaptation to different partnerships, we show that Seep-SRB1a partners of ANME-2c appear to lack nutritional auxotrophies, while the related Seep-SRB1a partners of a different methanotrophic archaeal lineage, ANME-2a, are missing the cobalamin synthesis pathway, suggesting that the Seep-SRB1a partners of ANME-2a may have a nutritional dependence on its partner. Together, our paired genomic analysis of AOM consortia highlights the specific adaptation and diversification of syntrophic SRB clades linked to their associated ANME lineages.

microbiology↗

bioRxiv2022

Evolutionary Diversification of Methanotrophic Ca. Methanophagales (ANME-1) and Their Expansive Virome

Candidatus Methanophagales (ANME-1) is a major order-level clade of archaea responsible for methane removal in deep-sea sediments through the anaerobic oxidation of methane. Yet the extent of their diversity and factors which drive their dynamics and evolution remain poorly understood. Here, by sampling hydrothermal rocks and sediments, we expand their phylogenetic diversity and characterize a new deep-branching, thermophilic ANME-1 family, Candidatus Methanospirareceae (ANME-1c). They are phylogenetically closest to the short-chain-alkane oxidizers Candidatus Syntrophoarchaeales and Candidatus Alkanophagales, and encode ancestral features including a methyl coenzyme M reductase chaperone McrD and a hydrogenase complex. Global phylogeny and near-complete genomes clarified that the debated hydrogen metabolism within ANME-1 is an ancient trait that was vertically inherited but differentially lost during lineage diversification. Our expanded genomic and metagenomic sampling allowed the discovery of viruses constituting 3 new orders and 16 new families that so far are exclusive to ANME-1 hosts. These viruses represent 4 major archaeal virus assemblages, characterized by tailless icosahedral, head-tailed, rod-shaped, and spindle-shaped virions, but display unique structural and replicative signatures. Exemplified by the analyses of thymidylate synthases that unveiled a virus-mediated ancestral process of host gene displacement, this expansive ANME-1 virome carries a large gene repertoire that can influence their hosts across different timescales. Our study thus puts forth an emerging evolutionary continuum between anaerobic methane and short-chain-alkane oxidizers and opens doors for exploring the impacts of viruses on the dynamics and evolution of the anaerobic methane-driven ecosystems.

microbiology↗

bioRxiv2021

Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features which separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well-distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

microbiology↗

bioRxiv2021

Microbial community of recently discovered Auka vent field sheds light on vent biogeography and evolutionary history of thermophily

Hydrothermal vents have been key to our understanding of the limits of life, and the metabolic and phylogenetic diversity of thermophilic organisms. Here we used environmental metagenomics combined with analysis of physico-chemical data and 16S rRNA amplicons to characterize the diversity, temperature optima, and biogeographic distribution of sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California, the deepest known hydrothermal vent field in the Pacific Ocean. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 1/3 of the currently known phylum diversity, showing the microbial community in Auka hydrothermal sediments is highly diverse. Large scale 16S rRNA amplicon screening of 227 sediment samples across the vent field indicates that the MAGs are largely representative of the microbial community. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota (Tharpobacteria) suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison of the average nucleotide identity of the Auka MAGs with MAGs from the Guaymas Basin vent field, found 400 km to the Northwest, revealed a remarkable 20% species-level overlap between vent sites, suggestive of long-distance species transfer and sediment colonization. An adapted version of a recently developed model for predicting optimal growth temperature to the Auka and Guaymas MAGs indicates several of these uncultured microorganisms could grow at temperatures exceeding the currently known upper limit of life. Extending this analysis to reference data shows that thermophily is a trait that has evolved frequently among Bacteria and Archaea. Combined, our results show that Auka vent field offers new perspectives on our understanding of hydrothermal vent microbiology.

genomics↗

bioRxiv2020

Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy

Archaeal anaerobic methanotrophs ( ANME) and sulfate-reducing Deltaproteobacteria ( SRB) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA Illumina iTag amplicon libraries prepared from Costa Rica sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g and diazotrophic activity within ANME-2b/SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain-reaction (HCR-) FISH targeting nifH transcripts and by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b/SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

microbiology↗