Isolation and genomic analysis of "Metallumcola ferriviriculae" MK1, a Gram-positive, Fe(III)-reducing bacterium from the Soudan Underground Mine, an iron-rich Martian analog site.

The Soudan Underground Mine State Park, found in the Vermilion Iron Range in northern Minnesota, provides access to a ~ 2.7 billion-year-old banded iron formation. Exploratory boreholes drilled between 1958 and 1962 on the 27th level (713 m underground) of the mine intersect calcium and iron-rich brines that have recently been subject to metagenomic analysis and microbial enrichments. Using concentrated brine samples pumped from a borehole depth of up to 55 m, a novel Gram-positive bacterium was enriched under anaerobic, acetate-oxidizing, and Fe(III) citrate-reducing conditions. The isolated bacterium, designated strain MK1, is non-motile, rod-shaped, spore-forming, anaerobic, and mesophilic, with a growth range between 24°C and 30°C. The complete circular MK1 genome was found to be 3,720,236 bp and encodes 25 putative multiheme cytochromes, including homologs to inner membrane cytochromes in the Gram-negative bacterium Geobacter sulfurreducens and cytoplasmic membrane and periplasmic cytochromes in the Gram-positive bacterium Thermincola potens. However, MK1 does not encode homologs of the peptidoglycan (CwcA) and cell surface-associated (OcwA) multiheme cytochromes proposed to be required by T. potens to perform extracellular electron transfer. The 16S rRNA gene sequence of MK1 indicates that its closest related isolate is Desulfitibacter alkalitolerans strain sk.kt5 (91% sequence identity), which places MK1 in a novel genus within the Desulfitibacteraceae family and Moorellales order. Within the Moorellales order, only Calderihabitans maritimus strain KKC1 has been reported to reduce Fe(III), and only D. alkalitolerans can also grow in temperatures below 40°C. Thus, MK1 represents a novel species within a novel genus, for which we propose the name "Metallumcola ferriviriculae" strain MK1, and provides a unique opportunity to study a cytochrome-rich, mesophilic, Gram-positive, spore-forming Fe(III)-reducing bacterium.IMPORTANCEThe Soudan Underground Mine State Park gives access to understudied regions of the deep terrestrial subsurface that potentially predate the Great Oxidation Event. Studying organisms that have been relatively unperturbed by surface conditions for as long as 2.7 billion years may give us a window into ancient life before oxygen dominated the planet. Additionally, studying microbes from anoxic and iron-rich environments can help us better understand the requirements of life in analogous environments, such as on Mars. The isolation and characterization of "Metallumcola ferriviriculae" strain MK1 give us insights into a novel genus and species that is distinct both from its closest related isolates and from iron reducers characterized to date. "M. ferriviriculae" strain MK1 may also act as a model organism to study how the processes of sporulation and germination are affected by insoluble extracellular acceptors, as well as the impact of spores in the deep terrestrial biosphere.

Distinct microbial nitrogen cycling processes in the deepest part of the ocean.

The Mariana Trench (MT) is the deepest part of the ocean on Earth. Previous studies have described the microbial community structures and functional potential in the seawater and surface sediment of MT. Still, the metabolic features and adaptation strategies of the microorganisms involved in nitrogen cycling processes are poorly understood. In this study, comparative metagenomic approaches were used to study microbial nitrogen cycling in three MT habitats, including hadal seawater [9,600-10,500 m below sea level (mbsl)], surface sediments [0-46 cm below seafloor (cmbsf) at a water depth between 7,143 and 8,638 mbsl], and deep sediments (200-306 cmbsf at a water depth of 8,300 mbsl). We identified five new nitrite-oxidizing bacteria (NOB) lineages that had adapted to the oligotrophic MT slope sediment, via their CO2 fixation capability through the reductive tricarboxylic acid (rTCA) or Calvin-Benson-Bassham (CBB) cycle; an anammox bacterium might perform aerobic respiration and utilize sedimentary carbohydrates for energy generation because it contains genes encoding type A cytochrome c oxidase and complete glycolysis pathway. In seawater, abundant alkane-oxidizing Ketobacter species can fix inert N2 released from other denitrifying and/or anammox bacteria. This study further expands our understanding of microbial life in the largely unexplored deepest part of the ocean. IMPORTANCE: The metabolic features and adaptation strategies of the nitrogen cycling microorganisms in the deepest part of the ocean are largely unknown. This study revealed that anammox bacteria might perform aerobic respiration in response to nutrient limitation or O2 fluctuations in the Mariana Trench sediments. Meanwhile, an abundant alkane-oxidizing Ketobacter species could fix N2 in hadal seawater. This study provides new insights into the roles of hadal microorganisms in global nitrogen biogeochemical cycles. It substantially expands our understanding of the microbial life in the largely unexplored deepest part of the ocean.

Species-resolved, single-cell respiration rates reveal dominance of sulfate reduction in a deep continental subsurface ecosystem.

Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~103 cells mL-1) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers including Candidatus Desulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell-1 h-1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L-1 d-1, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable to Ca. Desulforudis audaxviator.

Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community.

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10 mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

Cold seep formation from salt diapir-controlled deep biosphere oases.

Deep sea cold seeps are sites where hydrogen sulfide, methane, and other hydrocarbon-rich fluids vent from the ocean floor. They are an important component of Earth's carbon cycle in which subsurface hydrocarbons form the energy source for highly diverse benthic micro- and macro-fauna in what is otherwise vast and spartan sea scape. Passive continental margin cold seeps are typically attributed to the migration of hydrocarbons generated from deeply buried source rocks. Many of these seeps occur over salt tectonic provinces, where the movement of salt generates complex fault systems that can enable fluid migration or create seals and traps associated with reservoir formation. The elevated advective heat transport of the salt also produces a chimney effect directly over these structures. Here, we provide geophysical and geochemical evidence that the salt chimney effect in conjunction with diapiric faulting drives a subsurface groundwater circulation system that brings dissolved inorganic carbon, nutrient-rich deep basinal fluids, and potentially overlying seawater onto the crests of deeply buried salt diapirs. The mobilized fluids fuel methanogenic archaea locally enhancing the deep biosphere. The resulting elevated biogenic methane production, alongside the upward heat-driven fluid transport, represents a previously unrecognized mechanism of cold seep formation and regulation.

Hydrogeological controls on microbial activity and habitability in the Precambrian continental crust.

Earth's deep continental subsurface is a prime setting to study the limits of life's relationship with environmental conditions and habitability. In Precambrian crystalline rocks worldwide, deep ancient groundwaters in fracture networks are typically oligotrophic, highly saline, and locally inhabited by low-biomass communities in which chemolithotrophic microorganisms may dominate. Periodic opening of new fractures can lead to penetration of surface water and/or migration of fracture fluids, both of which may trigger changes in subsurface microbial composition and activity. These hydrogeological processes and their impacts on subsurface communities may play a significant role in global cycles of key elements in the crust. However, to date, considerable uncertainty remains on how subsurface microbial communities may respond to these changes in hydrogeochemical conditions. To address this uncertainty, the biogeochemistry of Thompson mine (Manitoba, Canada) was investigated. Compositional and isotopic analyses of fracture waters collected here at ~1 km below land surface revealed different extents of mixing between subsurface brine and (paleo)meteoric waters. To investigate the effects this mixing may have had on microbial communities, the Most Probable Number technique was applied to test community response for a total of 13 different metabolisms. The results showed that all fracture waters were dominated by viable heterotrophic microorganisms which can utilize organic materials associated with aerobic/facultative anaerobic processes, sulfate reduction, or fermentation. Where mixing between subsurface brines and (paleo)meteoric waters occurs, the communities demonstrate higher cell densities and increased viable functional potentials, compared to the most saline sample. This study therefore highlights the connection between hydrogeologic heterogeneity and the heterogeneity of subsurface ecosystems in the crystalline rocks, and suggests that hydrogeology can have a considerable impact on the scope and scale of subsurface microbial communities on Earth and potentially beyond.

Deep subsurface microbial life in impact-altered Late Paleozoic granitoid rocks from the Chicxulub impact crater.

In 2016, IODP-ICDP Expedition 364 recovered an 829-meter-long core within the peak ring of the Chicxulub impact crater (Yucatán, Mexico), allowing us to investigate the post-impact recovery of the heat-sterilized deep continental microbial biosphere at the impact site. We recently reported increased cell biomass in the impact suevite, which was deposited within the first few hours of the Cenozoic, and that the overall microbial communities differed significantly between the suevite and the other main core lithologies (i.e., the granitic basement and the overlying Early Eocene marine sediments; Cockell et al., 2021). However, only seven rock intervals were previously analyzed from the geologically heterogenic and impact-deformed 587-m-long granitic core section below the suevite interval. Here, we used 16S rRNA gene profiling to study the microbial community composition in 45 intervals including (a) 31 impact-shocked granites, (b) 7 non-granitic rocks (i.e., consisting of suevite and impact melt rocks intercalated into the granites during crater formation and strongly serpentinized pre-impact sub-volcanic, ultramafic basanite/dolerite), and (c) 7 cross-cut mineral veins of anhydride and silica. Most recovered microbial taxa resemble those found in hydrothermal systems. Spearman correlation analysis confirmed that the borehole temperature, which gradually increased from 47 to 69°C with core depth, significantly shaped a subset of the vertically stratified modern microbial community composition in the granitic basement rocks. However, bacterial communities differed significantly between the impoverished shattered granites and nutrient-enriched non-granite rocks, even though both lithologies were at similar depths and temperatures. Furthermore, Spearman analysis revealed a strong correlation between the microbial communities and bioavailable chemical compounds and suggests the presence of chemolithoautotrophs, which most likely still play an active role in metal and sulfur cycling. These results indicate that post-impact microbial niche separation has also occurred in the granitic basement lithologies, as previously shown for the newly formed lithologies. Moreover, our data suggest that the impact-induced geochemical boundaries continue to shape the modern-day deep biosphere in the granitic basement underlying the Chicxulub crater.

First shotgun metagenomics study of Juan de Fuca deep-sea sediments reveals distinct microbial communities above, within, between, and below sulfate methane transition zones.

The marine deep subsurface is home to a vast microbial ecosystem, affecting biogeochemical cycles on a global scale. One of the better-studied deep biospheres is the Juan de Fuca (JdF) Ridge, where hydrothermal fluid introduces oxidants into the sediment from below, resulting in two sulfate methane transition zones (SMTZs). In this study, we present the first shotgun metagenomics study of unamplified DNA from sediment samples from different depths in this stratified environment. Bioinformatic analyses showed a shift from a heterotrophic, Chloroflexota-dominated community above the upper SMTZ to a chemolithoautotrophic Proteobacteria-dominated community below the secondary SMTZ. The reintroduction of sulfate likely enables respiration and boosts active cells that oxidize acetate, iron, and complex carbohydrates to degrade dead biomass in this low-abundance, low-diversity environment. In addition, analyses showed many proteins of unknown function as well as novel metagenome-assembled genomes (MAGs). The study provides new insights into microbial communities in this habitat, enabled by an improved DNA extraction protocol that allows a less biased view of taxonomic composition and metabolic activities, as well as uncovering novel taxa. Our approach presents the first successful attempt at unamplified shotgun sequencing samples from beyond 50 meters below the seafloor and opens new ways for capturing the true diversity and functional potential of deep-sea sediments.

Cell-specific rates of sulfate reduction and fermentation in the sub-seafloor biosphere.

Microorganisms in subsurface sediments live from recalcitrant organic matter deposited thousands or millions of years ago. Their catabolic activities are low, but the deep biosphere is of global importance due to its volume. The stability of deeply buried sediments provides a natural laboratory where prokaryotic communities that live in steady state with their environments can be studied over long time scales. We tested if a balance is established between the flow of energy, the microbial community size, and the basal power requirement needed to maintain cells in sediments buried meters below the sea floor. We measured rates of carbon oxidation by sulfate reduction and counted the microbial cells throughout ten carefully selected sediment cores with ages from years to millions of years. The rates of carbon oxidation were converted to power (J s-1 i.e., Watt) using the Gibbs free energy of the anaerobic oxidation of complex organic carbon. We separated energy dissipation by fermentation from sulfate reduction. Similarly, we separated the community into sulfate reducers and non-sulfate reducers based on the dsrB gene, so that sulfate reduction could be related to sulfate reducers. We found that the per-cell sulfate reduction rate was stable near 10-2 fmol C cell-1 day-1 right below the zone of bioturbation and did not decrease with increasing depth and sediment age. The corresponding power dissipation rate was 10-17 W sulfate-reducing cell-1. The cell-specific power dissipation of sulfate reducers in old sediments was similar to the slowest growing anaerobic cultures. The energy from mineralization of organic matter that was not dissipated by sulfate reduction was distributed evenly to all cells that did not possess the dsrB gene, i.e., cells operationally defined as fermenting. In contrast to sulfate reducers, the fermenting cells had decreasing catabolism as the sediment aged. A vast difference in power requirement between fermenters and sulfate reducers caused the microbial community in old sediments to consist of a minute fraction of sulfate reducers and a vast majority of fermenters.

Clay-associated microbial communities and their relevance for a nuclear waste repository in the Opalinus Clay rock formation.

Microorganisms are known to be natural agents of biocorrosion and mineral transformation, thereby potentially affecting the safety of deep geological repositories used for high-level nuclear waste storage. To better understand how resident microbial communities of the deep terrestrial biosphere may act on mineralogical and geochemical characteristics of insulating clays, we analyzed their structure and potential metabolic functions, as well as site-specific mineralogy and element composition from the dedicated Mont Terri underground research laboratory, Switzerland. We found that the Opalinus Clay formation is mainly colonized by Alphaproteobacteria, Firmicutes, and Bacteroidota, which are known for corrosive biofilm formation. Potential iron-reducing bacteria were predominant in comparison to methanogenic archaea and sulfate-reducing bacteria. Despite microbial communities in Opalinus Clay being in majority homogenous, site-specific mineralogy and geochemistry conditions have selected for subcommunities that display metabolic potential for mineral dissolution and transformation. Our findings indicate that the presence of a potentially low-active mineral-associated microbial community must be further studied to prevent effects on the repository's integrity over the long term.

The potential role of subseafloor fungi in driving the biogeochemical cycle of nitrogen under anaerobic conditions.

Fungi represent the dominant eukaryotic group of organisms in anoxic marine sedimentary ecosystems, ranging from a few centimeters to ~ 2.5 km below seafloor. However, little is known about how fungi can colonize anaerobic subseafloor environments for tens of millions of years and whether they play a role in elemental biogeochemical cycles. Based on metabolite detection, isotope tracer and gene analysis, we examined the anaerobic nitrogen conversion pathways of 19 fungal species (40 strains) isolated from1.3 to 2.5 km coal-bearing sediments below seafloor. Our results show for the first time that almost all fungi possess anaerobic denitrification, dissimilatory nitrate reduction to ammonium (DNRA), and nitrification pathways, but not anaerobic ammonium oxidation (anammox). Moreover, the distribution of fungi with different nitrogen-conversion abilities in subseafloor sediments was mainly determined by in situ temperature, CaCO3, and inorganic carbon contents. These findings suggest that fungi have multiple nitrogen transformation processes to cope with their requirements for a variety of nitrogen sources in nutrient deficient anaerobic subseafloor sedimentary environments.

An improved method for intracellular DNA (iDNA) recovery from terrestrial environments.

The simultaneous extraction of intracellular DNA (iDNA) and extracellular DNA (eDNA) can help to separate the living in situ community (represented by iDNA) from background DNA that originated both from past communities and from allochthonous sources. As iDNA and eDNA extraction protocols require separating cells from the sample matrix, their DNA yields are generally lower than direct methods that lyse the cells within the sample matrix. We, therefore, tested different buffers with and without adding a detergent mix (DM) in the extraction protocol to improve the recovery of iDNA from surface and subsurface samples that covered a variety of terrestrial environments. The combination of a highly concentrated sodium phosphate buffer plus DM significantly improved iDNA recovery for almost all tested samples. Additionally, the combination of sodium phosphate and EDTA improved iDNA recovery in most of the samples and even allowed the successful extraction of iDNA from extremely low-biomass iron-bearing rock samples taken from the deep biosphere. Based on our results, we recommend using a protocol with sodium phosphate in combination with either a DM (NaP 300 mM + DM) or EDTA (NaP + EDTA 300 mM). Furthermore, for studies that rely on the eDNA pool, we recommend using buffers solely based on sodium phosphate because the addition of EDTA or a DM resulted in a decrease in eDNA for most of the tested samples. These improvements can help reduce community bias in environmental studies and contribute to better characterizations of both modern and past ecosystems.

Enrichment of rare methanogenic Archaea shows their important ecological role in natural high-CO2 terrestrial subsurface environments.

Introduction: Long-term stability of underground CO2 storage is partially affected by microbial activity but our knowledge of these effects is limited, mainly due to a lack of sites. A consistently high flux of mantle-derived CO2 makes the Eger Rift in the Czech Republic a natural analogue to underground CO2 storage. The Eger Rift is a seismically active region and H2 is produced abiotically during earthquakes, providing energy to indigenous microbial communities. Methods: To investigate the response of a microbial ecosystem to high levels of CO2 and H2, we enriched microorganisms from samples from a 239.5 m long drill core from the Eger Rift. Microbial abundance, diversity and community structure were assessed using qPCR and 16S rRNA gene sequencing. Enrichment cultures were set up with minimal mineral media and H2/CO2 headspace to simulate a seismically active period with elevated H2. Results and discussion: Methane headspace concentrations in the enrichments indicated that active methanogens were almost exclusively restricted to enrichment cultures from Miocene lacustrine deposits (50-60 m), for which we observed the most significant growth. Taxonomic assessment showed microbial communities in these enrichments to be less diverse than those with little or no growth. Active enrichments were especially abundant in methanogens of the taxa Methanobacterium and Methanosphaerula. Concurrent to the emergence of methanogenic archaea, we also observed sulfate reducers with the metabolic ability to utilize H2 and CO2, specifically the genus Desulfosporosinus, which were able to outcompete methanogens in several enrichments. Low microbial abundance and a diverse non-CO2 driven microbial community, similar to that in drill core samples, also reflect the inactivity in these cultures. Significant growth of sulfate reducing and methanogenic microbial taxa, which make up only a small fraction of the total microbial community, emphasize the need to account for rare biosphere taxa when assessing the metabolic potential of microbial subsurface populations. The observation that CO2 and H2-utilizing microorganisms could only be enriched from a narrow depth interval suggests that factors such as sediment heterogeneity may also be important. This study provides new insight on subsurface microbes under the influence of high CO2 concentrations, similar to those found in CCS sites.

Zonation of the active methane-cycling community in deep subsurface sediments of the Peru trench.

The production and anaerobic oxidation of methane (AOM) by microorganisms is widespread in organic-rich deep subseafloor sediments. Yet, the organisms that carry out these processes remain largely unknown. Here we identify members of the methane-cycling microbial community in deep subsurface, hydrate-containing sediments of the Peru Trench by targeting functional genes of the alpha subunit of methyl coenzyme M reductase (mcrA). The mcrA profile reveals a distinct community zonation that partially matches the zonation of methane oxidizing and -producing activity inferred from sulfate and methane concentrations and carbon-isotopic compositions of methane and dissolved inorganic carbon (DIC). McrA appears absent from sulfate-rich sediments that are devoid of methane, but mcrA sequences belonging to putatively methane-oxidizing ANME-1a-b occur from the zone of methane oxidation to several meters into the methanogenesis zone. A sister group of ANME-1a-b, referred to as ANME-1d, and members of putatively aceticlastic Methanothrix (formerly Methanosaeta) occur throughout the remaining methanogenesis zone. Analyses of 16S rRNA and mcrA-mRNA indicate that the methane-cycling community is alive throughout (rRNA to 230 mbsf) and active in at least parts of the sediment column (mRNA at 44 mbsf). Carbon-isotopic depletions of methane relative to DIC (-80 to -86‰) suggest mostly methane production by CO2 reduction and thus seem at odds with the widespread detection of ANME-1 and Methanothrix. We explain this apparent contradiction based on recent insights into the metabolisms of both ANME-1 and Methanothricaceae, which indicate the potential for methanogenetic growth by CO2 reduction in both groups.

Virus-Host Dynamics in Archaeal Groundwater Biofilms and the Associated Bacterial Community Composition.

Spatial and temporal distribution of lytic viruses in deep groundwater remains unexplored so far. Here, we tackle this gap of knowledge by studying viral infections of Altivir_1_MSI in biofilms dominated by the uncultivated host Candidatus Altiarchaeum hamiconexum sampled from deep anoxic groundwater over a period of four years. Using virus-targeted direct-geneFISH (virusFISH) whose detection efficiency for individual viral particles was 15%, we show a significant and steady increase of virus infections from 2019 to 2022. Based on fluorescence micrographs of individual biofilm flocks, we determined different stages of viral infections in biofilms for single sampling events, demonstrating the progression of infection of biofilms in deep groundwater. Biofilms associated with many host cells undergoing lysis showed a substantial accumulation of filamentous microbes around infected cells probably feeding off host cell debris. Using 16S rRNA gene sequencing across ten individual biofilm flocks from one sampling event, we determined that the associated bacterial community remains relatively constant and was dominated by sulfate-reducing members affiliated with Desulfobacterota. Given the stability of the virus-host interaction in these deep groundwater samples, we postulate that the uncultivated virus-host system described herein represents a suitable model system for studying deep biosphere virus-host interactions in future research endeavors.

Carbon metabolism and biogeography of candidate phylum "Candidatus Bipolaricaulota" in geothermal environments of Biga Peninsula, Turkey.

Terrestrial hydrothermal springs and aquifers are excellent sites to study microbial biogeography because of their high physicochemical heterogeneity across relatively limited geographic regions. In this study, we performed 16S rRNA gene sequencing and metagenomic analyses of the microbial diversity of 11 different geothermal aquifers and springs across the tectonically active Biga Peninsula (Turkey). Across geothermal settings ranging in temperature from 43 to 79°C, one of the most highly represented groups in both 16S rRNA gene and metagenomic datasets was affiliated with the uncultivated phylum "Candidatus Bipolaricaulota" (former "Ca. Acetothermia" and OP1 division). The highest relative abundance of "Ca. Bipolaricaulota" was observed in a 68°C geothermal brine sediment, where it dominated the microbial community, representing 91% of all detectable 16S rRNA genes. Correlation analysis of "Ca. Bipolaricaulota" operational taxonomic units (OTUs) with physicochemical parameters indicated that salinity was the strongest environmental factor measured associated with the distribution of this novel group in geothermal fluids. Correspondingly, analysis of 23 metagenome-assembled genomes (MAGs) revealed two distinct groups of "Ca. Bipolaricaulota" MAGs based on the differences in carbon metabolism: one group encoding the bacterial Wood-Ljungdahl pathway (WLP) for H2 dependent CO2 fixation is selected for at lower salinities, and a second heterotrophic clade that lacks the WLP that was selected for under hypersaline conditions in the geothermal brine sediment. In conclusion, our results highlight that the biogeography of "Ca. Bipolaricaulota" taxa is strongly correlated with salinity in hydrothermal ecosystems, which coincides with key differences in carbon acquisition strategies. The exceptionally high relative abundance of apparently heterotrophic representatives of this novel candidate Phylum in geothermal brine sediment observed here may help to guide future enrichment experiments to obtain representatives in pure culture.

The smallest in the deepest: the enigmatic role of viruses in the deep biosphere.

It is commonly recognized that viruses control the composition, metabolism, and evolutionary trajectories of prokaryotic communities, with resulting vital feedback on ecosystem functioning and nutrient cycling in a wide range of ecosystems. Although the deep biosphere has been estimated to be the largest reservoir for viruses and their prokaryotic hosts, the biology and ecology of viruses therein remain poorly understood. The deep virosphere is an enigmatic field of study in which many critical questions are still to be answered. Is the deep virosphere simply a repository for deeply preserved, non-functioning virus particles? Or are deep viruses infectious agents that can readily infect suitable hosts and subsequently shape microbial populations and nutrient cycling? Can the cellular content released by viral lysis, and even the organic structures of virions themselves, serve as the source of bioavailable nutrients for microbial activity in the deep biosphere as in other ecosystems? In this review, we synthesize our current knowledge of viruses in the deep biosphere and seek to identify topics with the potential for substantial discoveries in the future.