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To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNAtargeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

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 used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.

The α-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems 1 . This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods 2 . The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems 3 , 4 , 5 , which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study 6 , 7 . Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 µm 3 , places them among the smallest free-living cells in culture.

Abstract Identification of both prokaryotic and eukaryotic microorganisms in environmental samples is currently challenged by the need for additional sequencing to obtain separate 16S and 18S ribosomal RNA (rRNA) amplicons or the constraints imposed by “universal” primers. Organellar 16S rRNA sequences are amplified and sequenced along with prokaryote 16S rRNA and 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. The CABO-16S framework allows users to add custom sequences for further classification of underrepresented clades and can be easily updated with future releases of reference 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. CABO-16S highlights the benefit of combining and amending existing training sets when studying microorganisms in diverse environments. Graphical Abstract Graphical Abstract

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 amplicon libraries prepared from Costa Rica seep 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 within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH 4 and 15 N 2 revealed differences in cellular 15 N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15 N 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.

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 physicochemical data and 16S rRNA gene amplicons to characterize the sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 30% of those currently known, showing the microbial community in Auka hydrothermal sediments is highly diverse. 16S rRNA gene amplicon screening of 224 sediment samples across the vent field indicates that the MAGs retrieved from a single site are representative of the microbial community in the vent field sediments. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison between Auka MAGs and MAGs from Guaymas Basin revealed a remarkable 20% species-level overlap, suggestive of long-distance species transfer over 400 km and subsequent sediment colonization. Optimal growth temperature prediction on the Auka MAGs, and thousands of reference genomes, shows that thermophily is a trait that has evolved frequently. Taken together, our Auka vent field results offer new perspectives on our understanding of hydrothermal vent microbiology.

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A ( mcrA ), adenosine-5′-phosphosulfate reductase α-subunit ( aprA ), dinitrogenase reductase ( nifH )), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no 15 NH 4 + assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N 2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria . However, nifH expression was methane-dependent. 15 N 2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, 15 N 2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced 15 N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.

At methane seeps worldwide, syntrophic anaerobic methane-oxidizing archaea and sulfate-reducing bacteria promote carbonate precipitation and rock formation, acting as methane and carbon sinks. Although maintenance of anaerobic oxidation of methane (AOM) within seep carbonates has been documented, its reactivation upon methane exposure remains uncertain. Surface-associated microbes may metabolize sulfide from AOM, maintain carbonate anoxia, contribute to carbonate dissolution, and support higher trophic levels; however, these communities are poorly described. We provide insights into microbial diversity, metabolism, activity, and resiliency within and on seep carbonates through amplicon and metagenomic sequencing, incubations, and non-canonical amino acid tagging combined with fluorescence in situ hybridization (BONCAT-FISH). Ca. Methanophaga (ANME-1) dominated the carbonate interiors in active and low activity seeps, co-occurring with Ca. Desulfaltia as main sulfate reducer, potentially a new syntrophic partner in AOM. Single-cell BONCAT-FISH revealed variability in ANME-1 activity, suggesting potential dormancy in carbonates from low activity seep sites. However, incubations with carbonates from low activity seeps (≥24 months) showed exponential AOM reactivation (~44-day doubling), suggesting these carbonates retain the potential as long-term methane sinks under dynamic seepage conditions. Surface-associated microbial communities were heterogeneous and distinct from the carbonate interior and other seep habitats. Anaerobic methane-oxidizing biofilms and sulfide-oxidizing mats were associated with carbonates with high and intermediate AOM rates potentially influencing carbonate precipitation/dissolution. Shared aerobic methanotrophs between carbonate surfaces and invertebrates indicated carbonate surfaces may represent animal epibiont reservoirs. Recovered particulate methane monooxygenases included both aerobic methanotrophs and divergent forms associated with the Methylophagaceae, suggesting a new function in this group.

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during [smallcapital l]-alanine induced germination via terbium ion (Tb³⁺)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295±19 spores mL⁻¹ and 369±36 spores mL⁻¹, respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27±2 spores mL⁻¹. The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10³±6.7 x 10² cells mL⁻¹. Culturing attempts yielded 2 CFU mL⁻¹ (4°C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.

Eukaryotic genomes are known to have garnered innovations from both archaeal and bacterial domains but the sequence of events that led to the complex gene repertoire of eukaryotes is largely unresolved. Here, through the enrichment of hydrothermal vent microorganisms, we recovered two circularized genomes of Heimdallarchaeum species that belong to an Asgard archaea clade phylogenetically closest to eukaryotes. These genomes reveal diverse mobile elements, including an integrative viral genome that bidirectionally replicates in a circular form and aloposons, transposons that encode the 5,000 amino acid-sized proteins Otus and Ephialtes . Heimdallaechaeal mobile elements have garnered various genes from bacteria and bacteriophages, likely playing a role in shuffling functions across domains. The number of archaea- and bacteria-related genes follow strikingly different scaling laws in Asgard archaea, exhibiting a genome size-dependent ratio and a functional division resembling the bacteria- and archaea-derived gene repertoire across eukaryotes. Bacterial gene import has thus likely been a continuous process unaltered by eukaryogenesis and scaled up through genome expansion. Our data further highlight the importance of viewing eukaryogenesis in a pan-Asgard context, which led to the proposal of a conceptual framework, that is, the Heimdall nucleation–decentralized innovation–hierarchical import model that accounts for the emergence of eukaryotic complexity. The recovery of two circularized genomes of the Heimdallarchaeum species from hydrothermal vent enrichment cultures reveals that these Asgard archaea carry diverse mobile genetic elements, such as an integrative viral genome and aloposons. These mobile genetic elements contain several bacteria- and phage-derived genes, modulating the shuffling of information between bacteria and archaea, and potentially influencing eukaryogenesis.