Sulfur cycling and host-virus interactions in Aquificales-dominated biofilms from Yellowstone's hottest ecosystems.

Modern linkages among magmatic, geochemical, and geobiological processes provide clues about the importance of thermophiles in the origin of biogeochemical cycles. The aim of this study was to identify the primary chemoautotrophs and host-virus interactions involved in microbial colonization and biogeochemical cycling at sublacustrine, vapor-dominated vents that represent the hottest measured ecosystems in Yellowstone National Park (~140 °C). Filamentous microbial communities exposed to extreme thermal and geochemical gradients were sampled using a remotely operated vehicle and subjected to random metagenome sequencing and microscopic analyses. Sulfurihydrogenibium (phylum Aquificae) was the predominant lineage (up to 84% relative abundance) detected at vents that discharged high levels of dissolved H2, H2S, and CO2. Metabolic analyses indicated carbon fixation by Sulfurihydrogenibium spp. was powered by the oxidation of reduced sulfur and H2, which provides organic carbon for heterotrophic community members. Highly variable Sulfurihydrogenibium genomes suggested the importance of intra-population diversity under extreme environmental and viral pressures. Numerous lytic viruses (primarily unclassified taxa) were associated with diverse archaea and bacteria in the vent community. Five circular dsDNA uncultivated virus genomes (UViGs) of ~40 kbp length were linked to the Sulfurihydrogenibium metagenome-assembled genome (MAG) by CRISPR spacer matches. Four UViGs contained consistent genome architecture and formed a monophyletic cluster with the recently proposed Pyrovirus genus within the Caudovirales. Sulfurihydrogenibium spp. also contained CRISPR arrays linked to plasmid DNA with genes for a novel type IV filament system and a highly expressed ß-barrel porin. A diverse suite of transcribed secretion systems was consistent with direct microscopic analyses, which revealed an extensive extracellular matrix likely critical to community structure and function. We hypothesize these attributes are fundamental to the establishment and survival of microbial communities in highly turbulent, extreme-gradient environments.

The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.

IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element­guided activity), with strong potential for developing as biotechnologies.

Draft Genome Sequence of Methanothermobacter thermautotrophicus WHS, a Thermophilic Hydrogenotrophic Methanogen from Washburn Hot Springs in Yellowstone National Park, USA.

A thermophilic methanogen was enriched in coculture from Washburn Hot Springs (Yellowstone National Park, USA), grown on carbon dioxide and hydrogen, and subsequently sequenced. The reconstructed 1.65-Mb genome sequence for Methanothermobacter thermautotrophicus WHS contributes to our understanding of hydrogenotrophic, CO2-reducing methanogenesis in geothermal ecosystems.

Roadmap for naming uncultivated Archaea and Bacteria.

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

Physiology, Metabolism, and Fossilization of Hot-Spring Filamentous Microbial Mats.

The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87-98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.

Wide diversity of methane and short-chain alkane metabolisms in uncultured archaea.

Methanogenesis is an ancient metabolism of key ecological relevance, with direct impact on the evolution of Earth's climate. Recent results suggest that the diversity of methane metabolisms and their derivations have probably been vastly underestimated. Here, by probing thousands of publicly available metagenomes for homologues of methyl-coenzyme M reductase complex (MCR), we have obtained ten metagenome-assembled genomes (MAGs) belonging to potential methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea. Five of these MAGs represent under-sampled (Verstraetearchaeota, Methanonatronarchaeia, ANME-1 and GoM-Arc1) or previously genomically undescribed (ANME-2c) archaeal lineages. The remaining five MAGs correspond to lineages that are only distantly related to previously known methanogens and span the entire archaeal phylogeny. Comprehensive comparative annotation substantially expands the metabolic diversity and energy conservation systems of MCR-bearing archaea. It also suggests the potential existence of a yet uncharacterized type of methanogenesis linked to short-chain alkane/fatty acid oxidation in a previously undescribed class of archaea ('Candidatus Methanoliparia'). We redefine a common core of marker genes specific to methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea, and propose a possible scenario for the evolutionary and functional transitions that led to the emergence of such metabolic diversity.

Co-occurring genomic capacity for anaerobic methane and dissimilatory sulfur metabolisms discovered in the Korarchaeota.

Phylogenetic and geological evidence supports the hypothesis that life on Earth originated in thermal environments and conserved energy through methanogenesis or sulfur reduction. Here we describe two populations of the deeply rooted archaeal phylum Korarchaeota, which were retrieved from the metagenome of a circumneutral, suboxic hot spring that contains high levels of sulfate, sulfide, methane, hydrogen and carbon dioxide. One population is closely related to 'Candidatus Korarchaeum cryptofilum OPF8', while the more abundant korarchaeote, 'Candidatus Methanodesulfokores washburnensis', contains genes that are necessary for anaerobic methane and dissimilatory sulfur metabolisms. Phylogenetic and ancestral reconstruction analyses suggest that methane metabolism originated in the Korarchaeota, whereas genes for dissimilatory sulfite reduction were horizontally transferred to the Korarchaeota from the Firmicutes. Interactions among enzymes involved in both metabolisms could facilitate exergonic, sulfite-dependent, anaerobic oxidation of methane to methanol; alternatively, 'Ca. M. washburnensis' could conduct methanogenesis and sulfur reduction independently. Metabolic reconstruction suggests that 'Ca. M. washburnensis' is a mixotroph, capable of amino acid uptake, assimilation of methane-derived carbon and/or CO2 fixation by archaeal type III-b RuBisCO for scavenging ribose carbon. Our findings link anaerobic methane metabolism and dissimilatory sulfur reduction within a single deeply rooted archaeal population and have implications for the evolution of these traits throughout the Archaea.

Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community.

Interactions among microbial community members can lead to emergent properties, such as enhanced productivity, stability, and robustness. Iron-oxide mats in acidic (pH 2-4), high-temperature (> 65 °C) springs of Yellowstone National Park contain relatively simple microbial communities and are well-characterized geochemically. Consequently, these communities are excellent model systems for studying the metabolic activity of individual populations and key microbial interactions. The primary goals of the current study were to integrate data collected in situ with in silico calculations across process-scales encompassing enzymatic activity, cellular metabolism, community interactions, and ecosystem biogeochemistry, as well as to predict and quantify the functional limits of autotroph-heterotroph interactions. Metagenomic and transcriptomic data were used to reconstruct carbon and energy metabolisms of an important autotroph (Metallosphaera yellowstonensis) and heterotroph (Geoarchaeum sp. OSPB) from the studied Fe(III)-oxide mat communities. Standard and hybrid elementary flux mode and flux balance analyses of metabolic models predicted cellular- and community-level metabolic acclimations to simulated environmental stresses, respectively. In situ geochemical analyses, including oxygen depth-profiles, Fe(III)-oxide deposition rates, stable carbon isotopes and mat biomass concentrations, were combined with cellular models to explore autotroph-heterotroph interactions important to community structure-function. Integration of metabolic modeling with in situ measurements, including the relative population abundance of autotrophs to heterotrophs, demonstrated that Fe(III)-oxide mat communities operate at their maximum total community growth rate (i.e. sum of autotroph and heterotroph growth rates), as opposed to net community growth rate (i.e. total community growth rate subtracting autotroph consumed by heterotroph), as predicted from the maximum power principle. Integration of multiscale data with ecological theory provides a basis for predicting autotroph-heterotroph interactions and community-level cellular organization.

Marsarchaeota are an aerobic archaeal lineage abundant in geothermal iron oxide microbial mats.

The discovery of archaeal lineages is critical to our understanding of the universal tree of life and evolutionary history of the Earth. Geochemically diverse thermal environments in Yellowstone National Park provide unprecedented opportunities for studying archaea in habitats that may represent analogues of early Earth. Here, we report the discovery and characterization of a phylum-level archaeal lineage proposed and herein referred to as the 'Marsarchaeota', after the red planet. The Marsarchaeota contains at least two major subgroups prevalent in acidic, microaerobic geothermal Fe(III) oxide microbial mats across a temperature range from ~50-80 °C. Metagenomics, single-cell sequencing, enrichment culturing and in situ transcriptional analyses reveal their biogeochemical role as facultative aerobic chemoorganotrophs that may also mediate the reduction of Fe(III). Phylogenomic analyses of replicate assemblies corresponding to two groups of Marsarchaeota indicate that they branch between the Crenarchaeota and all other major archaeal lineages. Transcriptomic analyses of several Fe(III) oxide mat communities reveal that these organisms were actively transcribing two different terminal oxidase complexes in situ and genes comprising an F420-dependent butanal catabolism. The broad distribution of Marsarchaeota in geothermal, microaerobic Fe(III) oxide mats suggests that similar habitat types probably played an important role in the evolution of archaea.

Occurrence and expression of novel methyl-coenzyme M reductase gene (mcrA) variants in hot spring sediments.

Recent discoveries have shown that the marker gene for anaerobic methane cycling (mcrA) is more widespread in the Archaea than previously thought. However, it remains unclear whether novel mcrA genes associated with the Bathyarchaeota and Verstraetearchaeota are distributed across diverse environments. We examined two geochemically divergent but putatively methanogenic regions of Yellowstone National Park to investigate whether deeply-rooted archaea possess and express novel mcrA genes in situ. Small-subunit (SSU) rRNA gene analyses indicated that Bathyarchaeota were predominant in seven of ten sediment layers, while the Verstraetearchaeota and Euryarchaeota occurred in lower relative abundance. Targeted amplification of novel mcrA genes suggested that diverse taxa contribute to alkane cycling in geothermal environments. Two deeply-branching mcrA clades related to Bathyarchaeota were identified, while highly abundant verstraetearchaeotal mcrA sequences were also recovered. In addition, detection of SSU rRNA and mcrA transcripts from one hot spring suggested that predominant Bathyarchaeota were also active, and that methane cycling genes are expressed by the Euryarchaeota, Verstraetearchaeota, and an unknown lineage basal to the Bathyarchaeota. These findings greatly expand the diversity of the key marker gene for anaerobic alkane cycling and outline the need for greater understanding of the functional capacity and phylogenetic affiliation of novel mcrA variants.

Integration of Metagenomic and Stable Carbon Isotope Evidence Reveals the Extent and Mechanisms of Carbon Dioxide Fixation in High-Temperature Microbial Communities.

Although the biological fixation of CO2 by chemolithoautotrophs provides a diverse suite of organic compounds utilized by chemoorganoheterotrophs as a carbon and energy source, the relative amounts of autotrophic C in chemotrophic microbial communities are not well-established. The extent and mechanisms of CO2 fixation were evaluated across a comprehensive set of high-temperature, chemotrophic microbial communities in Yellowstone National Park by combining metagenomic and stable 13C isotope analyses. Fifteen geothermal sites representing three distinct habitat types (iron-oxide mats, anoxic sulfur sediments, and filamentous "streamer" communities) were investigated. Genes of the 3-hydroxypropionate/4-hydroxybutyrate, dicarboxylate/4-hydroxybutyrate, and reverse tricarboxylic acid CO2 fixation pathways were identified in assembled genome sequence corresponding to the predominant Crenarchaeota and Aquificales observed across this habitat range. Stable 13C analyses of dissolved inorganic and organic C (DIC, DOC), and possible landscape C sources were used to interpret the 13C content of microbial community samples. Isotope mixing models showed that the minimum fractions of autotrophic C in microbial biomass were >50% in the majority of communities analyzed. The significance of CO2 as a C source in these communities provides a foundation for understanding community assembly and succession, and metabolic linkages among early-branching thermophilic autotrophs and heterotrophs.

Stoichiometric modelling of assimilatory and dissimilatory biomass utilisation in a microbial community.

Assimilatory and dissimilatory utilisation of autotroph biomass by heterotrophs is a fundamental mechanism for the transfer of nutrients and energy across trophic levels. Metagenome data from a tractable, thermoacidophilic microbial community in Yellowstone National Park was used to build an in silico model to study heterotrophic utilisation of autotroph biomass using elementary flux mode analysis and flux balance analysis. Assimilatory and dissimilatory biomass utilisation was investigated using 29 forms of biomass-derived dissolved organic carbon (DOC) including individual monomer pools, individual macromolecular pools and aggregate biomass. The simulations identified ecologically competitive strategies for utilizing DOC under conditions of varying electron donor, electron acceptor or enzyme limitation. The simulated growth environment affected which form of DOC was the most competitive use of nutrients; for instance, oxygen limitation favoured utilisation of less reduced and fermentable DOC while carbon-limited environments favoured more reduced DOC. Additionally, metabolism was studied considering two encompassing metabolic strategies: simultaneous versus sequential use of DOC. Results of this study bound the transfer of nutrients and energy through microbial food webs, providing a quantitative foundation relevant to most microbial ecosystems.

The distribution, diversity and function of predominant Thermoproteales in high-temperature environments of Yellowstone National Park.

High-temperature environments (> 70°C) contain diverse and abundant members of the crenarchaeal order Thermoproteales. However, a comprehensive study of the distribution and function of diverse members of this group across different habitat types has not been conducted. Consequently, the goals of this study were to determine the distribution of different Thermoproteales genera across geochemically distinct geothermal habitats of Yellowstone National Park, and to identify key functional attributes of major genera that correlate with environmental parameters. Curated sequence assemblies belonging to five genera were characterized in replicate samples of 11 high-temperature communities ranging in pH from 3 to 9. Thermocladium, Vulcanisaeta and Caldivirga spp. were the primary Thermoproteales populations present in low pH (pH < 5) habitats, whereas Thermoproteus populations were found in mildly-acidic (pH 5-6) sulfur sediments, and Pyrobaculum populations were confined to higher pH (pH > 6) sulfur sediments and/or filamentous 'streamer' communities. Metabolic reconstruction and comparative genomics among assemblies show that these populations are primarily chemoorganotrophs that utilize different electron acceptors depending on geochemical conditions. The presence of potential CO2 fixation pathways in some Thermoproteales populations appears to be linked with NiFe hydrogenases, which combined with high levels of H2 in many sulfidic systems, may provide the energy required to fix inorganic C.

Novel, Deep-Branching Heterotrophic Bacterial Populations Recovered from Thermal Spring Metagenomes.

Thermal spring ecosystems are a valuable resource for the discovery of novel hyperthermophilic Bacteria and Archaea, and harbor deeply-branching lineages that provide insight regarding the nature of early microbial life. We characterized bacterial populations in two circumneutral (pH ~8) Yellowstone National Park thermal (T ~80°C) spring filamentous "streamer" communities using random metagenomic DNA sequence to investigate the metabolic potential of these novel populations. Four de novo assemblies representing three abundant, deeply-branching bacterial phylotypes were recovered. Analysis of conserved phylogenetic marker genes indicated that two of the phylotypes represent separate groups of an uncharacterized phylum (for which we propose the candidate phylum name "Pyropristinus"). The third new phylotype falls within the proposed Calescamantes phylum. Metabolic reconstructions of the "Pyropristinus" and Calescamantes populations showed that these organisms appear to be chemoorganoheterotrophs and have the genomic potential for aerobic respiration and oxidative phosphorylation via archaeal-like V-type, and bacterial F-type ATPases, respectively. A survey of similar phylotypes (>97% nt identity) within 16S rRNA gene datasets suggest that the newly described organisms are restricted to terrestrial thermal springs ranging from 70 to 90°C and pH values of ~7-9. The characterization of these lineages is important for understanding the diversity of deeply-branching bacterial phyla, and their functional role in high-temperature circumneutral "streamer" communities.

Geochemistry and Mixing Drive the Spatial Distribution of Free-Living Archaea and Bacteria in Yellowstone Lake.

Yellowstone Lake, the largest subalpine lake in the United States, harbors great novelty and diversity of Bacteria and Archaea. Size-fractionated water samples (0.1-0.8, 0.8-3.0, and 3.0-20 µm) were collected from surface photic zone, deep mixing zone, and vent fluids at different locations in the lake by using a remotely operated vehicle (ROV). Quantification with real-time PCR indicated that Bacteria dominated free-living microorganisms with Bacteria/Archaea ratios ranging from 4037:1 (surface water) to 25:1 (vent water). Microbial population structures (both Bacteria and Archaea) were assessed using 454-FLX sequencing with a total of 662,302 pyrosequencing reads for V1 and V2 regions of 16S rRNA genes. Non-metric multidimensional scaling (NMDS) analyses indicated that strong spatial distribution patterns existed from surface to deep vents for free-living Archaea and Bacteria in the lake. Along with pH, major vent-associated geochemical constituents including CH4, CO2, H2, DIC (dissolved inorganic carbon), DOC (dissolved organic carbon), SO4 (2-), O2 and metals were likely the major drivers for microbial population structures, however, mixing events occurring in the lake also impacted the distribution patterns. Distinct Bacteria and Archaea were present among size fractions, and bigger size fractions included particle-associated microbes (> 3 µm) and contained higher predicted operational taxonomic unit richness and microbial diversities (genus level) than free-living ones (<0.8 µm). Our study represents the first attempt at addressing the spatial distribution of Bacteria and Archaea in Yellowstone Lake, and our results highlight the variable contribution of Archaea and Bacteria to the hydrogeochemical-relevant metabolism of hydrogen, carbon, nitrogen, and sulfur.

Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin, Yellowstone National Park.

The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with (13)C-formaldehyde. Isotopic analysis of incubation-derived CO2 and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.

Assembly and Succession of Iron Oxide Microbial Mat Communities in Acidic Geothermal Springs.

Biomineralized ferric oxide microbial mats are ubiquitous features on Earth, are common in hot springs of Yellowstone National Park (YNP, WY, USA), and form due to direct interaction between microbial and physicochemical processes. The overall goal of this study was to determine the contribution of different community members to the assembly and succession of acidic high-temperature Fe(III)-oxide mat ecosystems. Spatial and temporal changes in Fe(III)-oxide accretion and the abundance of relevant community members were monitored over 70 days using sterile glass microscope slides incubated in the outflow channels of two acidic geothermal springs (pH = 3-3.5; temperature = 68-75°C) in YNP. Hydrogenobaculum spp. were the most abundant taxon identified during early successional stages (4-40 days), and have been shown to oxidize arsenite, sulfide, and hydrogen coupled to oxygen reduction. Iron-oxidizing populations of Metallosphaera yellowstonensis were detected within 4 days, and reached steady-state levels within 14-30 days, corresponding to visible Fe(III)-oxide accretion. Heterotrophic archaea colonized near 30 days, and emerged as the dominant functional guild after 70 days and in mature Fe(III)-oxide mats (1-2 cm thick). First-order rate constants of Fe(III)-oxide accretion ranged from 0.046 to 0.05 day(-1), and in situ microelectrode measurements showed that the oxidation of Fe(II) is limited by the diffusion of O2 into the Fe(III)-oxide mat. The formation of microterracettes also implicated O2 as a major variable controlling microbial growth and subsequent mat morphology. The assembly and succession of Fe(III)-oxide mat communities follows a repeatable pattern of colonization by lithoautotrophic organisms, and the subsequent growth of diverse organoheterotrophs. The unique geochemical signatures and micromorphology of extant biomineralized Fe(III)-oxide mats are also useful for understanding other Fe(II)-oxidizing systems.

Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous 'streamer' community.

The candidate archaeal phylum 'Aigarchaeota' contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous 'streamer' community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus 'Calditenuis aerorheumensis' for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.