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Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water–rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

Many Archaea produce membrane-spanning glycerol dibiphytanyl glycerol tetraether (GDGTs) lipids that serve as unique biomarkers of past environments. These lipids can contain up to eight cyclopentane rings, where an increase in ring cyclization is generally associated with growth in more acidic, higher temperature, or more energy limited conditions. Recently the genes that encode GDGT ring synthases, grsAB, were identified and characterized in model thermoacidophiles Sulfolobus acidocaldarius and Saccharolobus solfataricus. However, the distribution and abundance of grs homologs across environments inhabited by these and related Archaea remains unknown. To address this, we examined the distribution of grs homologs in archaeal and bacterial cultivar genomes, single cell genomes, metagenomes, and metatranscriptomes from thermal springs across the planet, where temperature, pH, and geochemical data take at time of sampling. The relative abundance of grs in these microbial communities exhibits a strong negative correlation with pH, and weak positive correlation with temperature. Genomes and metagenome-assembled genomes (MAGs) from Archaea that encode two or more copies of grs are significantly more widespread in low pH springs. Homologs of grs were detected in MAGs from 12 archaeal classes, with the most well-represented being the Thermoproteia. Homologs of grs were also detected among several classes of uncultured Archaea, including the Korarchaeia, Bathyarchaeia, and Hadarchaeia. Several Nitrososphaeria MAGs had high copy numbers of grs (> 3), and the functional role of these copies cannot yet be explained. Notably, grs genes were also found in MAGs from the bacterial class Acidobacteria. Based on phylogenetic analyses, it is likely that Acidobacteria acquired these genes horizontally from Archaea. Broadly, our results highlight the key role of grs-catalyzed lipid cyclization in the diversification of Archaea in hot and acidic environments.

Metagenome assembled genomes (MAGs) and single amplified genomes (SAGs) affiliated with two distinct Methanobacterium lineages were recovered from subsurface fracture waters of the Samail Ophiolite, Sultanate of Oman. Lineage Type I was abundant in waters with circumneutral pH, whereas lineage Type II was abundant in hydrogen rich, hyperalkaline waters. Type I encoded proteins to couple hydrogen oxidation to CO2 reduction, typical of hydrogenotrophic methanogens. Surprisingly, Type II, which branched from the Type I lineage, lacked homologs of two key oxidative [NiFe]-hydrogenases. These functions were presumably replaced by formate dehydrogenases that oxidize formate to yield reductant and cytoplasmic CO2 via a pathway that was unique among characterized Methanobacteria, allowing cells to overcome CO2/oxidant limitation in high pH waters. This prediction was supported by microcosm-based radiotracer experiments that showed significant biological methane generation from formate, but not bicarbonate, in waters where the Type II lineage was detected in highest relative abundance. Phylogenetic analyses and variability in gene content suggested that recent and ongoing diversification of the Type II lineage was enabled by gene transfer, loss, and transposition. These data indicate that selection imposed by CO2/oxidant availability drove recent methanogen diversification into hyperalkaline waters that are heavily impacted by serpentinization.

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

Extremophiles exist among all three domains of life; however, physiological mechanisms for surviving harsh environmental conditions differ among Bacteria, Archaea and Eukarya. Consequently, we expect that domain-specific variation of diversity and community assembly patterns exist along environmental gradients in extreme environments. We investigated inter-domain community compositional differences along a high-elevation salinity gradient in the McMurdo Dry Valleys, Antarctica. Conductivity for 24 soil samples collected along the gradient ranged widely from 50 to 8355 µS cm

Little is known of how the confluence of subsurface and surface processes influences the assembly and habitability of hydrothermal ecosystems. To address this knowledge gap, the geochemical and microbial composition of a high-temperature, circumneutral hot spring in Yellowstone National Park was examined to identify the sources of solutes and their effect on the ecology of microbial inhabitants. Metagenomic analysis showed that populations comprising planktonic and sediment communities are archaeal dominated, are dependent on chemical energy (chemosynthetic), share little overlap in their taxonomic composition, and are differentiated by their inferred use of/tolerance to oxygen and mode of carbon metabolism. The planktonic community is dominated by putative aerobic/aerotolerant autotrophs, while the taxonomic composition of the sediment community is more evenly distributed and comprised of anaerobic heterotrophs. These observations are interpreted to reflect sourcing of the spring by anoxic, organic carbon-limited subsurface hydrothermal fluids and ingassing of atmospheric oxygen that selects for aerobic/aerotolerant organisms that have autotrophic capabilities in the water column. Autotrophy and consumption of oxygen by the planktonic community may influence the assembly of the anaerobic and heterotrophic sediment community. Support for this inference comes from higher estimated rates of genome replication in planktonic populations than sediment populations, indicating faster growth in planktonic populations. Collectively, these observations provide new insight into how mixing of subsurface waters and atmospheric oxygen create dichotomy in the ecology of hot spring communities and suggest that planktonic and sediment communities may have been less differentiated taxonomically and functionally prior to the rise of oxygen at ∼2.4 billion years ago (Gya). IMPORTANCE Understanding the source and availability of energy capable of supporting life in hydrothermal environments is central to predicting the ecology of microbial life on early Earth when volcanic activity was more widespread. Little is known of the substrates supporting microbial life in circumneutral to alkaline springs, despite their relevance to early Earth habitats. Using metagenomic and informatics approaches, water column and sediment habitats in a representative circumneutral hot spring in Yellowstone were shown to be dichotomous, with the former largely hosting aerobic/aerotolerant autotrophs and the latter primarily hosting anaerobic heterotrophs. This dichotomy is attributed to influx of atmospheric oxygen into anoxic deep hydrothermal spring waters. These results indicate that the ecology of microorganisms in circumneutral alkaline springs sourced by deep hydrothermal fluids was different prior to the rise of atmospheric oxygen ∼2.4 Gya, with planktonic and sediment communities likely to be less differentiated than contemporary circumneutral hot springs.

Trace metals have been an important ingredient for life throughout Earth’s history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles.

Decompressional boiling of ascending hydrothermal waters and separation into a vapor (gas) and a liquid phase drive extensive variation in the geochemical composition of hot spring waters. Yet little is known of how the process of phase separation influences the distribution of microbial metabolisms in springs. Here, we determined the variation in protein coding genes in 51 metagenomes from chemosynthetic hot spring communities that span geochemical gradients in Yellowstone National Park. The 51 metagenomes could be divided into 5 distinct groups that correspond to low and high temperatures and acidic and circumneutral/alkaline springs. A fifth group primarily comprised metagenomes from springs with moderate acidity and that are influenced by elevated volcanic gas input. Protein homologs putatively involved in the oxidation of sulfur compounds, a process that leads to acidification of spring waters, in addition to those involved in the reduction of sulfur compounds were enriched in metagenomes from acidic springs sourced by vapor phase gases. Metagenomes from springs with evidence for elevated volcanic gas input were enriched in protein homologs putatively involved in oxidation of those gases, including hydrogen and methane. Finally, metagenomes from circumneutral/alkaline springs sourced by liquid phase waters were enriched in protein homologs putatively involved in heterotrophy and respiration of oxidized nitrogen compounds and oxygen. These results indicate that the geological process of phase separation shapes the ecology of thermophilic communities through its influence on the availability of nutrients in the form of gases, solutes, and minerals. Microbial acidification of hot spring waters further influences the kinetic and thermodynamic stabilities of nutrients and their bioavailability. These data therefore provide an important framework to understand how geological processes have shaped the evolutionary history of chemosynthetic thermophiles and how these organisms, in turn, have shaped their geochemical environments.

Little is known about how the geological history of an environment shapes its physical and chemical properties and how these, in turn, influence the assembly of communities. Evening primrose (EP), a moderately acidic hot spring (pH 5.6, 77.4°C) in Yellowstone National Park (YNP), has undergone dramatic physicochemical change linked to seismic activity. Here, we show that this legacy of geologic change led to the development of an unusual sulphur-rich, anoxic chemical environment that supports a unique archaeal-dominated and anaerobic microbial community. Metagenomic sequencing and informatics analyses reveal that >96% of this community is supported by dissimilatory reduction or disproportionation of inorganic sulphur compounds, including a novel, deeply diverging sulphate-reducing thaumarchaeote. When compared to other YNP metagenomes, the inferred functions of EP populations were like those from sulphur-rich acidic springs, suggesting that sulphur may overprint the predominant influence of pH on the composition of hydrothermal communities. Together, these observations indicate that the dynamic geological history of EP underpins its unique geochemistry and biodiversity, emphasizing the need to consider the legacy of geologic change when describing processes that shape the assembly of communities.

Hydrogen (H2 ) is enriched in hot springs and can support microbial primary production. Using a series of geochemical proxies, a model to describe variable H2 concentrations in Yellowstone National Park (YNP) hot springs is presented. Interaction between water and crustal iron minerals yields H2 that partition into the vapour phase during decompressional boiling of ascending hydrothermal fluids. Variable vapour input leads to differences in H2 concentration among springs. Analysis of 50 metagenomes from a variety of YNP springs reveals that genes encoding oxidative hydrogenases are enriched in communities inhabiting springs sourced with vapour-phase gas. Three springs in the Smokejumper (SJ) area of YNP that are sourced with vapour-phase gas and with the most H2 in YNP were examined to determine the fate of H2 . SJ3 had the most H2 , the most 16S rRNA gene templates and the greatest abundance of culturable hydrogenotrophic and autotrophic cells of the three springs. Metagenomics and transcriptomics of SJ3 reveal a diverse community comprised of abundant populations expressing genes involved in H2 oxidation and carbon dioxide fixation. These observations suggest a link between geologic processes that generate and source H2 to hot springs and the distribution of organisms that use H2 to generate energy.

Hydration of ultramafic rock during the geologic process of serpentinization can generate reduced substrates that microorganisms may use to fuel their carbon and energy metabolisms. However, serpentinizing environments also place multiple constraints on microbial life by generating highly reduced hyperalkaline waters that are limited in dissolved inorganic carbon. To better understand how microbial life persists under these conditions, we performed geochemical measurements on waters from a serpentinizing environment and subjected planktonic microbial cells to metagenomic and physiological analyses. Metabolic potential inferred from metagenomes correlated with fluid type, and genes involved in anaerobic metabolisms were enriched in hyperalkaline waters. The abundance of planktonic cells and their rates of utilization of select single-carbon compounds were lower in hyperalkaline waters than alkaline waters. However, the ratios of substrate assimilation to dissimilation were higher in hyperalkaline waters than alkaline waters, which may represent adaptation to minimize energetic and physiologic stress imposed by highly reducing, carbon-limited conditions. Consistent with this hypothesis, estimated genome sizes and average oxidation states of carbon in inferred proteomes were lower in hyperalkaline waters than in alkaline waters. These data suggest that microorganisms inhabiting serpentinized waters exhibit a unique suite of physiological adaptations that allow for their persistence under these polyextremophilic conditions.

Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), and the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2 dehydrogenase), protoheme (AhbABCD), cytochrome c (Cyt c) (CcmCF), and iron storage/detoxification (Bfr, FtrA, and IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. The results indicate several prevalent but nonuniversal features, including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold, as well as its siroheme (and F430) derivatives. However, several early-diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited equivalent prevalences of [Fe-S] cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities, while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved. IMPORTANCE Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for cofactor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group. Variation in these characters was generally congruent with the phylogenetic placement of these genomes, indicating that variation in Fe/S usage and deployment has contributed to the diversification and ecology of these organisms. However, incongruency was observed among the distribution of cofactor biosynthesis pathways and known protein destinations for those cofactors, suggesting auxotrophy or yet-to-be-discovered pathways for cofactor biosynthesis.

Hot springs integrate hydrologic and geologic processes that vary over short- and long-term time scales. However, the influence of temporal hydrologic and geologic change on hot spring biodiversity is unknown. Here, we coordinated near-weekly, cross-seasonal (~140 days) geochemical and microbial community analyses of three widely studied hot springs with local precipitation data in Yellowstone National Park. One spring ('HFS') exhibited statistically significant, coupled microbial and geochemical variation across seasons that was associated with recent precipitation patterns. Two other spring communities, 'CP' and 'DS', exhibited minimal to no variation across seasons. Variability in the seasonal response of springs is attributed to differences in the timing and extent of aquifer recharge with oxidized near-surface water from precipitation. This influx of oxidized water is associated with changes in community composition, and in particular, the abundances of aerobic sulfide-/sulfur-oxidizers that can acidify waters. During sampling, a new spring formed after a period of heavy precipitation and its successional dynamics were also influenced by surface water recharge. Collectively, these results indicate that changes in short-term hydrology associated with precipitation can impact hot spring geochemistry and microbial biodiversity. These results point to potential susceptibility of certain hot springs and their biodiversity to sustained, longer-term hydrologic changes.

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understandingits molecular basis in such inhospitable conditions, given that such conditions lead to loss of structure and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments. We present the state of recent progress in extreme environmental genomics. We then present an overview of our current understanding of the biomolecular adaptation to extreme conditions. As our current and future understanding of biomolecular structure–function relationships in extremophiles requires methodologies adapted to extremes of pressure, temperature, and chemical composition, advances in instrumentation for probing biophysical properties under extreme conditions are presented. Finally, we briefly discuss possible future directions in extreme biophysics.

Correction to: Nature Microbiology https://doi.org/10.1038/s41564-020-0733-x , published online 8 June 2020. In the version of this Consensus Statement originally published, Pablo Yarza was mistakenly not included in the author list. Also, in Supplementary Table 1, Alexander Jaffe was missing from the list of endorsees. These errors have now been corrected and the updated Supplementary Table 1 is available online.

8 pages, 2 figures, 1 table, 2 boxes, supplementary information https://doi.org/10.1038/s41564-020-0733-x.-- Author Correction: Roadmap for naming uncultivated Archaea and Bacteria: n the version of this Consensus Statement originally published, Pablo Yarza was mistakenly not included in the author list. Also, in Supplementary Table 1, Alexander Jaffe was missing from the list of endorsees. These errors have now been corrected and the updated Supplementary Table 1 is available online, 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, Support for this effort was provided by the National Science Foundation (grant no. DEB-1841658). We extend deep appreciation to all who attended the Microbial Taxonomy 2018 seminars, the Microbial Systematics for the Next Decade workshop held in Hood River, Oregon, USA, in October 2018, and those that participated in subsequent, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Microbial mat communities are associated with extensive (∼700 km2) and morphologically variable carbonate structures, termed microbialites, in the hypersaline Great Salt Lake (GSL), Utah. However, whether the composition of GSL mat communities covaries with microbialite morphology and lake environment is unknown. Moreover, the potential adaptations that allow the establishment of these extensive mat communities at high salinity (14% to 17% total salts) are poorly understood. To address these questions, microbial mats were sampled from seven locations in the south arm of GSL representing different lake environments and microbialite morphologies. Despite the morphological differences, microbialite-associated mats were taxonomically similar and were dominated by the cyanobacterium Euhalothece and several heterotrophic bacteria. Metagenomic sequencing of a representative mat revealed Euhalothece and subdominant Thiohalocapsa populations that harbor the Calvin cycle and nitrogenase, suggesting they supply fixed carbon and nitrogen to heterotrophic bacteria. Fifteen of the next sixteen most abundant taxa are inferred to be aerobic heterotrophs and, surprisingly, harbor reaction center, rhodopsin, and/or bacteriochlorophyll biosynthesis proteins, suggesting aerobic photoheterotrophic (APH) capabilities. Importantly, proteins involved in APH are enriched in the GSL community relative to that in microbialite mat communities from lower salinity environments. These findings indicate that the ability to integrate light into energy metabolism is a key adaptation allowing for robust mat development in the hypersaline GSL. IMPORTANCE The earliest evidence of life on Earth is from organosedimentary structures, termed microbialites, preserved in 3.481-billion-year-old (Ga) rocks. Phototrophic microbial mats form in association with an ∼700-km2 expanse of morphologically diverse microbialites in the hypersaline Great Salt Lake (GSL), Utah. Here, we show taxonomically similar microbial mat communities are associated with morphologically diverse microbialites across the lake. Metagenomic sequencing reveals an abundance and diversity of autotrophic and heterotrophic taxa capable of harvesting light energy to drive metabolism. The unexpected abundance of and diversity in the mechanisms of harvesting light energy observed in GSL mat populations likely function to minimize niche overlap among coinhabiting taxa, provide a mechanism(s) to increase energy yield and osmotic balance during salt stress, and enhance fitness. Together, these physiological benefits promote the formation of robust mats that, in turn, influence the formation of morphologically diverse microbialite structures that can be imprinted in the rock record.

Large-scale environmental disturbances may impact both partners in coral host-Symbiodinium systems. Elucidation of the assembly patterns in such complex and interdependent communities may enable better prediction of environmental impacts across coral reef ecosystems. In this study, we investigated how the community composition and diversity of dinoflagellate symbionts in the genus Symbiodinium were distributed among 12 host species from six taxonomic orders (Actinaria, Alcyonacea, Miliolida, Porifera, Rhizostoma, Scleractinia) and in the reef water and sediments at Lizard Island, Great Barrier Reef before the 3(rd) Global Coral Bleaching Event. 454 pyrosequencing of the ITS2 region of Symbiodinium yielded 83 Operational Taxonomic Units (OTUs) at a 97% similarity cut-off. Approximately half of the Symbiodinium OTUs from reef water or sediments were also present in symbio. OTUs belonged to six clades (A-D, F-G), but community structure was uneven. The two most abundant OTUs (100% matches to types C1 and A3) comprised 91% of reads and OTU C1 was shared by all species. However, sequence-based analysis of these dominant OTUs revealed host species-specificity, suggesting that genetic similarity cut-offs of Symbiodinium ITS2 data sets need careful evaluation. Of the less abundant OTUs, roughly half occurred at only one site or in one species and the background Symbiodinium communities were distinct between individual samples. We conclude that sampling multiple host taxa with differing life history traits will be critical to fully understand the symbiont diversity of a given system and to predict coral ecosystem responses to environmental change and disturbance considering the differential stress response of the taxa within.

Serpentinite-hosted systems represent modern-day analogs of early Earth environments. In these systems, water-rock interactions generate highly alkaline and reducing fluids that can contain hydrogen, methane, and low-molecular-weight hydrocarbons-potent reductants capable of fueling microbial metabolism. In this study, we investigated the microbiota of Hakuba Happo hot springs (∼50°C; pH∼10.5-11), located in Nagano (Japan), which are impacted by the serpentinization process. Analysis of the 16S rRNA gene amplicon sequences revealed that the bacterial community comprises Nitrospirae (47%), "Parcubacteria" (19%), Deinococcus-Thermus (16%), and Actinobacteria (9%), among others. Notably, only 57 amplicon sequence variants (ASV) were detected, and fifteen of these accounted for 90% of the amplicons. Among the abundant ASVs, an early-branching, uncultivated actinobacterial clade identified as RBG-16-55-12 in the SILVA database was detected. Ten single-cell genomes (average pairwise nucleotide identity: 0.98-1.00; estimated completeness: 33-93%; estimated genome size: ∼2.3 Mb) that affiliated with this clade were obtained. Taxonomic classification using single copy genes indicates that the genomes belong to the actinobacterial class-level clade UBA1414 in the Genome Taxonomy Database. Based on metabolic pathway predictions, these actinobacteria are anaerobes, capable of glycolysis, dissimilatory nitrate reduction and CO2 fixation via the Wood-Ljungdahl (WL) pathway. Several other genomes within UBA1414 and two related class-level clades also encode the WL pathway, which has not yet been reported for the Actinobacteria phylum. For the Hakuba actinobacterium, the energy metabolism related to the WL pathway is likely supported by a combination of the Rnf complex, group 3b and 3d [NiFe]-hydrogenases, [FeFe]-hydrogenases, and V-type (H+/Na+ pump) ATPase. The genomes also harbor a form IV ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, also known as a RubisCO-like protein, and contain signatures of interactions with viruses, including clustered regularly interspaced short palindromic repeat (CRISPR) regions and several phage integrases. This is the first report and detailed genome analysis of a bacterium within the Actinobacteria phylum capable of utilizing the WL pathway. The Hakuba actinobacterium is a member of the clade UBA1414/RBG-16-55-12, formerly within the group "OPB41." We propose to name this bacterium 'Candidatus Hakubanella thermoalkaliphilus.'

Background [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. Methods To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. Results HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. Conclusions These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. General significance This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.

Biological nitrogen fixation via the activity of nitrogenase is one of the most important biological innovations, allowing for an increase in global productivity that eventually permitted the emergence of higher forms of life. The complex metalloenzyme termed nitrogenase contains complex iron-sulfur cofactors. Three versions of nitrogenase exist that differ mainly by the presence or absence of a heterometal at the active site metal cluster (either Mo or V). Mo-dependent nitrogenase is the most common while V-dependent or heterometal independent (Fe-only) versions are often termed alternative nitrogenases since they have apparent lower activities for N2 reduction and are expressed in the absence of Mo. Phylogenetic data indicates that biological nitrogen fixation emerged in an anaerobic, thermophilic ancestor of hydrogenotrophic methanogens and later diversified via lateral gene transfer into anaerobic bacteria, and eventually aerobic bacteria including Cyanobacteria. Isotopic evidence suggests that nitrogenase activity existed at 3.2 Ga, prior to the advent of oxygenic photosynthesis and rise of oxygen in the atmosphere, implying the presence of favorable environmental conditions for oxygen-sensitive nitrogenase to evolve. Following the proliferation of oxygenic phototrophs, diazotrophic organisms had to develop strategies to protect nitrogenase from oxygen inactivation and generate the right balance of low potential reducing equivalents and cellular energy for growth and nitrogen fixation activity. Here we review the fundamental advances in our understanding of biological nitrogen fixation in the context of the emergence, evolution, and taxonomic distribution of nitrogenase, with an emphasis placed on key events associated with its emergence and diversification from anoxic to oxic environments.

Little is known of how mixing of meteoric and geothermal fluids supports biodiversity in non-photosynthetic ecosystems. Here, we use metagenomic sequencing to investigate a chemosynthetic microbial community in a hot spring (SJ3) of Yellowstone National Park that exhibits geochemistry consistent with mixing of a reduced volcanic gas-influenced end member with an oxidized near-surface meteoric end member. SJ3 hosts an exceptionally diverse community with representatives from ~50% of known higher-order archaeal and bacterial lineages, including several divergent deep-branching lineages. A comparison of functional potential with other available chemosynthetic community metagenomes reveals similarly high diversity and functional potentials (i.e., incorporation of electron donors supplied by volcanic gases) in springs sourced by mixed fluids. Further, numerous closely related SJ3 populations harbor differentiated metabolisms that may function to minimize niche overlap, further increasing endemic diversity. We suggest that dynamic mixing of waters generated by subsurface and near-surface geological processes may play a key role in the generation and maintenance of chemosynthetic biodiversity in hydrothermal and other similar environments., Chemosynthetic microbial communities in hydrothermal environments receiving meteoric and geothermal fluids are understudied. Here, Colman et al. use metagenomics to study one such community from a hot spring at Yellowstone National Park, revealing exceptional biodiversity and unique functional potential.

The distribution of organisms in an environment is neither uniform nor random but is instead spatially patterned. The factors that control this patterning are complex and the underlying mechanisms are poorly understood. Soil microbes are critical to ecosystem function but exhibit highly complex distributions and community dynamics due in large part to the scale-dependent effects of environmental heterogeneity. To better understand the impact of environmental heterogeneity on the distribution of soil microbes, we sequenced the 16S rRNA gene from bacterial communities in the microbe-dominated polar desert ecosystem of the McMurdo Dry Valleys (MDV), Antarctica. Significant differences in key edaphic variables and alpha diversity were observed among the three lake basins of the Taylor Valley (Kruskal–Wallis; pH: χ2 = 68.89, P < 0.001, conductivity: χ2 = 35.03, P < 0.001, observed species: χ2 = 7.98, P = 0.019 and inverse Simpson: χ2 = 18.52, P < 0.001) and each basin supported distinctive microbial communities (ANOSIM R = 0.466, P = 0.001, random forest ratio of 14.1). However, relationships between community structure and edaphic characteristics were highly variable and contextual, ranging in magnitude and direction across regional, basin, and local scales. Correlations among edaphic factors (pH and soil conductivity) and the relative abundance of specific phyla were most pronounced along local environmental gradients in the Lake Fryxell basin where Acidobacteria, Bacteroidetes, and Proteobacteria declined while Deinococcus–Thermus and Gemmatimonadetes increased with soil conductivity (all P < 0.1). Species richness was most strongly related to the soil conductivity gradient present within this study system. We suggest that the relative importance of pH versus soil conductivity in structuring microbial communities is related to the length of edaphic gradients and the spatial scale of sampling. These results highlight the importance of conducting studies over large ranges of key environmental gradients and across multiple spatial scales to assess the influence of environmental heterogeneity on the composition and diversity of microbial communities.

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe3+ ) or oxygen (O2 ) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H2 ) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe3+ or O2 grown cells. During growth on S° or Fe3+ with H2 as an electron donor, the amount of CO2 assimilated into biomass decreased when cultures were provided with acetate. The addition of CO2 to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H2 /Fe3+ grown cells but had no effect on H2 /S° grown cells. In acetate/Fe3+ grown cells, the presence of H2 decreased the amount of acetate mineralized as CO2 in cultures compared to those without H2 . These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H2 to cultures overcomes this limitation and alters heterotrophic metabolism.

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O 2 )-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N 2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O 2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N 2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation. IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O 2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.

The discovery of oceanic black (and white) smokers revolutionized our understanding of mid-ocean ridges and led to the recognition of new organisms and ecosystems. Continental smokers, defined here to include a broad range of carbonic springs, hot springs, and fumaroles that vent mantle-derived fluids in continental settings, exhibit many of the same processes of heat and mass transfer and ecosystem niche differentiation. Helium isotope (3He/4He) analyses indicate that widespread mantle degassing is taking place in the western U.S.A., and that variations in mantle helium values correlate best with low seismic-velocity domains in the mantle and lateral contrasts in mantle velocity rather than crustal parameters such as GPS, proximity to volcanoes, crustal velocity, or composition. Microbial community analyses indicate that these springs can host novel microorganisms. A targeted analysis of four springs in New Mexico yield the first published occurrence of chemolithoautotrophic Zetaproteobacteria in a continental setting. These observations lead to two linked hypotheses: that mantle-derived volatiles transit through conduits in extending continental lithosphere preferentially above and at the edges of mantle low velocity domains. High CO2 and other constituents ultimately derived from mantle volatiles drive water–rock interactions and heterogeneous fluid mixing that help structure diverse and distinctive microbial communities.

[FeFe]- and [NiFe]-hydrogenases are two classes of enzymes that couple the reversible reduction of protons to the production of hydrogen gas. While these enzymes have unique taxonomic distributions and differences in their structure, function, maturation, and evolutionary history, they nevertheless share important similarities and may both be of critical importance for photobiological hydrogen production. Here, the structural and functional diversity of both types of hydrogenases are discussed in light of their phylogenetic and metabolic contexts. Overviews of the mechanisms of hydrogen activation at the respective active sites are given and provide insight into the role of the surrounding protein coordination sphere in catalysis and tuning reactivity. The distinct maturation pathways of both the [FeFe]- and [NiFe]-hydrogenases are described, revealing unique processes for assembling diatomic carbon monoxide and cyanide ligands onto Fe atoms, a key feature of the active sites that facilitates hydrogen activation during turnover conditions. Finally, a discussion of bifurcating [FeFe]- and [NiFe]-hydrogenases sheds light on this recently characterized group of enzymes that may hold a profound advantage at energetically efficient hydrogen production.

Oxygen-dependent microbial oxidation of sulfur compounds leads to the acidification of natural waters. How acidophiles and their acidic habitats evolved, however, is largely unknown. Using 16S rRNA gene abundance and composition data from 72 hot springs in Yellowstone National Park, Wyoming, we show that hyperacidic (pH