Metal-oxide precipitation influences microbiome structure in hyporheic zones receiving acid rock drainage.

Streams impacted by historic mining activity are characterized by acidic pH, unique microbial communities, and abundant metal-oxide precipitation, all of which can influence groundwater-surface water exchange. We investigate how metal-oxide precipitates and hyporheic mixing mediate the composition of microbial communities in two streams receiving acid-rock and mine drainage near Silverton, Colorado, USA. A large, neutral pH hyporheic zone facilitated the precipitation of metal particles/colloids in hyporheic porewaters. A small, low pH hyporheic zone, limited by the presence of a low-permeability, iron-oxyhydroxide layer known as ferricrete, led to the formation of steep geochemical gradients and high dissolved-metal concentrations. To determine how these two hyporheic systems influence microbiome composition, we installed well clusters and deployed in situ microcosms in each stream to sample porewaters and sediments for 16S rRNA gene sequencing. Results indicated that distinct hydrogeochemical conditions were present above and below the ferricrete in the low pH system. A positive feedback loop may be present in the low pH stream where microbially mediated precipitation of iron-oxides contributes to additional clogging of hyporheic pore spaces, separating abundant, iron-oxidizing bacteria (Gallionella spp.) above the ferricrete from rare, low-abundance bacteria below the ferricrete. Metal precipitates and colloids that formed in the neutral pH hyporheic zone were associated with a more diverse phylogenetic community of nonmotile, nutrient-cycling bacteria that may be transported through hyporheic pore spaces. In summary, biogeochemical conditions influence, and are influenced by, hyporheic mixing, which mediates the distribution of micro-organisms and, thus, the cycling of metals in streams receiving acid-rock and mine drainage. IMPORTANCE: In streams receiving acid-rock and mine drainage, the abundant precipitation of iron minerals can alter how groundwater and surface water mix along streams (in what is known as the "hyporheic zone") and may shape the distribution of microbial communities. The findings presented here suggest that neutral pH streams with large, well-mixed hyporheic zones may harbor and transport diverse microorganisms attached to particles/colloids through hyporheic pore spaces. In acidic streams where metal oxides clog pore spaces and limit hyporheic exchange, iron-oxidizing bacteria may dominate and phylogenetic diversity becomes low. The abundance of iron-oxidizing bacteria in acid mine drainage streams has the potential to contribute to additional clogging of hyporheic pore spaces and the accumulation of toxic metals in the hyporheic zone. This research highlights the dynamic interplay between hydrology, geochemistry, and microbiology at the groundwater-surface water interface of acid mine drainage streams.

Seasonal variation in the biological succession of marine diatoms over 316L stainless steel in a coastal environment of Chile.

Characterizing seasonal changes in diatom community profiles in coastal environments is scarce worldwide. Despite diatoms being prevalent in microfouling, their role in microbially influenced corrosion of metallic materials remains poorly understood. This study reports the effect of seasonal variations on the settlement of marine diatoms and corrosion of 316 L stainless steel surfaces exposed to Chilean coastal seawater. Electron microscopy imaging revealed a diverse assembly of diatoms, exhibiting pronounced differences at genus level between summer and winter seasons, with a significant delay in diatom settlement during winter. Electrochemical measurements indicated an active role of diatoms in increasing corrosion current during biofilm development. While the final diatom composition was similar irrespective of the season, the analyses of diatom assemblages over time differed, showing faster colonization when silicate and nitrate were available. This study lays the foundation for future research on the dominant season-specific genera of diatoms to unveil the microbial interactions that could contribute to corrosion and to evaluate their potential as bioindicators for alternative surveillance strategies.

Intact polar lipidome and membrane adaptations of microbial communities inhabiting serpentinite-hosted fluids.

The generation of hydrogen and reduced carbon compounds during serpentinization provides sustained energy for microorganisms on Earth, and possibly on other extraterrestrial bodies (e.g., Mars, icy satellites). However, the geochemical conditions that arise from water-rock reaction also challenge the known limits of microbial physiology, such as hyperalkaline pH, limited electron acceptors and inorganic carbon. Because cell membranes act as a primary barrier between a cell and its environment, lipids are a vital component in microbial acclimation to challenging physicochemical conditions. To probe the diversity of cell membrane lipids produced in serpentinizing settings and identify membrane adaptations to this environment, we conducted the first comprehensive intact polar lipid (IPL) biomarker survey of microbial communities inhabiting the subsurface at a terrestrial site of serpentinization. We used an expansive, custom environmental lipid database that expands the application of targeted and untargeted lipodomics in the study of microbial and biogeochemical processes. IPLs extracted from serpentinite-hosted fluid communities were comprised of >90% isoprenoidal and non-isoprenoidal diether glycolipids likely produced by archaeal methanogens and sulfate-reducing bacteria. Phospholipids only constituted ~1% of the intact polar lipidome. In addition to abundant diether glycolipids, betaine and trimethylated-ornithine aminolipids and glycosphingolipids were also detected, indicating pervasive membrane modifications in response to phosphate limitation. The carbon oxidation state of IPL backbones was positively correlated with the reduction potential of fluids, which may signify an energy conservation strategy for lipid synthesis. Together, these data suggest microorganisms inhabiting serpentinites possess a unique combination of membrane adaptations that allow for their survival in polyextreme environments. The persistence of IPLs in fluids beyond the presence of their source organisms, as indicated by 16S rRNA genes and transcripts, is promising for the detection of extinct life in serpentinizing settings through lipid biomarker signatures. These data contribute new insights into the complexity of lipid structures generated in actively serpentinizing environments and provide valuable context to aid in the reconstruction of past microbial activity from fossil lipid records of terrestrial serpentinites and the search for biosignatures elsewhere in our solar system.

Metapangenomic investigation provides insight into niche differentiation of methanogenic populations from the subsurface serpentinizing environment, Samail Ophiolite, Oman.

Serpentinization reactions produce highly reduced waters that have hyperalkaline pH and that can have high concentrations of H2 and CH4. Putatively autotrophic methanogenic archaea have been identified in the subsurface waters of the Samail Ophiolite, Sultanate of Oman, though the strategies to overcome hyperalkaline pH and dissolved inorganic carbon limitation remain to be fully understood. Here, we recovered metagenome assembled genomes (MAGs) and applied a metapangenomic approach to three different Methanobacterium populations to assess habitat-specific functional gene distribution. A Type I population was identified in the fluids with neutral pH, while a Type II and "Mixed" population were identified in the most hyperalkaline fluids (pH 11.63). The core genome of all Methanobacterium populations highlighted potential DNA scavenging techniques to overcome phosphate or nitrogen limitation induced by environmental conditions. With particular emphasis on the Mixed and Type II population found in the most hyperalkaline fluids, the accessory genomes unique to each population reflected adaptation mechanisms suggesting lifestyles that minimize niche overlap. In addition to previously reported metabolic capability to utilize formate as an electron donor and generate intracellular CO2, the Type II population possessed genes relevant to defense against antimicrobials and assimilating potential osmoprotectants to provide cellular stability. The accessory genome of the Mixed population was enriched in genes for multiple glycosyltransferases suggesting reduced energetic costs by adhering to mineral surfaces or to other microorganisms, and fostering a non-motile lifestyle. These results highlight the niche differentiation of distinct Methanobacterium populations to circumvent the challenges of serpentinization impacted fluids through coexistence strategies, supporting our ability to understand controls on methanogenic lifestyles and adaptations within the serpentinizing subsurface fluids of the Samail Ophiolite.

Subsurface biogeochemical cycling of nitrogen in the actively serpentinizing Samail Ophiolite, Oman.

Nitrogen (N) is an essential element for life. N compounds such as ammonium ( NH 4 + ) may act as electron donors, while nitrate ( NO 3 - ) and nitrite ( NO 2 - ) may serve as electron acceptors to support energy metabolism. However, little is known regarding the availability and forms of N in subsurface ecosystems, particularly in serpentinite-hosted settings where hydrogen (H2) generated through water-rock reactions promotes habitable conditions for microbial life. Here, we analyzed N and oxygen (O) isotope composition to investigate the source, abundance, and cycling of N species within the Samail Ophiolite of Oman. The dominant dissolved N species was dependent on the fluid type, with Mg2+- HCO 3 - type fluids comprised mostly of NO 3 - , and Ca2+-OH- fluids comprised primarily of ammonia (NH3). We infer that fixed N is introduced to the serpentinite aquifer as NO 3 - . High concentrations of NO 3 - (>100 µM) with a relict meteoric oxygen isotopic composition (δ18O ~ 22‰, Δ17O ~ 6‰) were observed in shallow aquifer fluids, indicative of NO 3 - sourced from atmospheric deposition (rainwater NO 3 - : δ18O of 53.7‰, Δ17O of 16.8‰) mixed with NO 3 - produced in situ through nitrification (estimated endmember δ18O and Δ17O of ~0‰). Conversely, highly reacted hyperalkaline fluids had high concentrations of NH3 (>100 µM) with little NO 3 - detectable. We interpret that NH3 in hyperalkaline fluids is a product of NO 3 - reduction. The proportionality of the O and N isotope fractionation (18ε / 15ε) measured in Samail Ophiolite NO 3 - was close to unity (18ε / 15ε ~ 1), which is consistent with dissimilatory NO 3 - reduction with a membrane-bound reductase (NarG); however, abiotic reduction processes may also be occurring. The presence of genes commonly involved in N reduction processes (narG, napA, nrfA) in the metagenomes of biomass sourced from aquifer fluids supports potential biological involvement in the consumption of NO 3 - . Production of NH 4 + as the end-product of NO 3 - reduction via dissimilatory nitrate reduction to ammonium (DNRA) could retain N in the subsurface and fuel nitrification in the oxygenated near surface. Elevated bioavailable N in all sampled fluids indicates that N is not likely limiting as a nutrient in serpentinites of the Samail Ophiolite.

Spatial and temporal dynamics at an actively silicifying hydrothermal system.

Steep Cone Geyser is a unique geothermal feature in Yellowstone National Park (YNP), Wyoming, actively gushing silicon-rich fluids along outflow channels possessing living and actively silicifying microbial biomats. To assess the geomicrobial dynamics occurring temporally and spatially at Steep Cone, samples were collected at discrete locations along one of Steep Cone's outflow channels for both microbial community composition and aqueous geochemistry analysis during field campaigns in 2010, 2018, 2019, and 2020. Geochemical analysis characterized Steep Cone as an oligotrophic, surface boiling, silicious, alkaline-chloride thermal feature with consistent dissolved inorganic carbon and total sulfur concentrations down the outflow channel ranging from 4.59 ± 0.11 to 4.26 ± 0.07 mM and 189.7 ± 7.2 to 204.7 ± 3.55 µM, respectively. Furthermore, geochemistry remained relatively stable temporally with consistently detectable analytes displaying a relative standard deviation <32%. A thermal gradient decrease of ~55°C was observed from the sampled hydrothermal source to the end of the sampled outflow transect (90.34°C ± 3.38 to 35.06°C ± 7.24). The thermal gradient led to temperature-driven divergence and stratification of the microbial community along the outflow channel. The hyperthermophile Thermocrinis dominates the hydrothermal source biofilm community, and the thermophiles Meiothermus and Leptococcus dominate along the outflow before finally giving way to more diverse and even microbial communities at the end of the transect. Beyond the hydrothermal source, phototrophic taxa such as Leptococcus, Chloroflexus, and Chloracidobacterium act as primary producers for the system, supporting heterotrophic growth of taxa such as Raineya, Tepidimonas, and Meiothermus. Community dynamics illustrate large changes yearly driven by abundance shifts of the dominant taxa in the system. Results indicate Steep Cone possesses dynamic outflow microbial communities despite stable geochemistry. These findings improve our understanding of thermal geomicrobiological dynamics and inform how we can interpret the silicified rock record.

An untargeted exometabolomics approach to characterize dissolved organic matter in groundwater of the Samail Ophiolite.

The process of serpentinization supports life on Earth and gives rise to the habitability of other worlds in our Solar System. While numerous studies have provided clues to the survival strategies of microbial communities in serpentinizing environments on the modern Earth, characterizing microbial activity in such environments remains challenging due to low biomass and extreme conditions. Here, we used an untargeted metabolomics approach to characterize dissolved organic matter in groundwater in the Samail Ophiolite, the largest and best characterized example of actively serpentinizing uplifted ocean crust and mantle. We found that dissolved organic matter composition is strongly correlated with both fluid type and microbial community composition, and that the fluids that were most influenced by serpentinization contained the greatest number of unique compounds, none of which could be identified using the current metabolite databases. Using metabolomics in conjunction with metagenomic data, we detected numerous products and intermediates of microbial metabolic processes and identified potential biosignatures of microbial activity, including pigments, porphyrins, quinones, fatty acids, and metabolites involved in methanogenesis. Metabolomics techniques like the ones used in this study may be used to further our understanding of life in serpentinizing environments, and aid in the identification of biosignatures that can be used to search for life in serpentinizing systems on other worlds.

Optode-based chemical imaging of laboratory burned soil reveals millimeter-scale heterogeneous biogeochemical responses.

Soil spatial responses to fire are unclear. Using optical chemical sensing with planar 'optodes', pH and dissolved O2 concentration were tracked spatially with a resolution of 360 µm per pixel for 72 h after burning soil in the laboratory with a butane torch (∼1300 °C) and then sprinkling water to simulate a postfire moisture event. Imaging data from planar optodes correlated with microbial activity (quantified via RNA transcripts). Post-fire and post-wetting, soil pH increased throughout the entire ∼13 cm × 17 cm × 20 cm rectangular cuboid of sandy loam soil. Dissolved O2 concentrations were not impacted until the application of water postfire. pH and dissolved O2 both negatively correlated (p < 0.05) with relative transcript expression for galactose metabolism, the degradation of aromatic compounds, sulfur metabolism, and narH. Additionally, dissolved O2 negatively correlated (p < 0.05) with the relative activity of carbon fixation pathways in Bacteria and Archaea, amoA/amoB, narG, nirK, and nosZ. nifH was not detected in any samples. Only amoB and amoC correlated with depth in soil (p < 0.05). Results demonstrate that postfire soils are spatially complex on a mm scale and that using optode-based chemical imaging as a chemical navigator for RNA transcript sampling is effective.

Deep-branching acetogens in serpentinized subsurface fluids of Oman.

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.

Statistical Learning and Uncommon Soil Microbiota Explain Biogeochemical Responses after Wildfire.

Wildfires are a perennial event globally, and the biogeochemical underpinnings of soil responses at relevant spatial and temporal scales are unclear. Soil biogeochemical processes regulate plant growth and nutrient losses that affect water quality, yet the response of soil after variable intensity fire is difficult to explain and predict. To address this issue, we examined two wildfires in Colorado, United States, across the first and second postfire years and leveraged statistical learning (SL) to predict and explain biogeochemical responses. We found that SL predicts biogeochemical responses in soil after wildfire with surprising accuracy. Of the 13 biogeochemical analytes analyzed in this study, 9 are best explained with a hybrid microbiome + biogeochemical SL model. Biogeochemical-only models best explain 3 features, and 1 feature is explained equally well with the hybrid and biogeochemical-only models. In some cases, microbiome-only SL models are also effective (such as predicting NH4+). Whenever a microbiome component is employed, selected features always involve uncommon soil microbiota (i.e., the "rare biosphere" [existing at <1% mean relative abundance]). Here, we demonstrate that SL paired with DNA sequence and biogeochemical data predicts environmental features in postfire soils, although this approach could likely be applied to any biogeochemical system. IMPORTANCE Soil biogeochemical processes are critical to plant growth and water quality and are substantially disturbed by wildfire. However, soil responses to fire are difficult to predict. To address this issue, we developed a large environmental data set that tracks postfire changes in soil and used statistical learning (SL) to build models that exploit complex data to make predictions about biogeochemical responses. Here, we show that SL depends upon uncommon microbiota in soil (the "rare biosphere") to make surprisingly accurate predictions about soil biogeochemical responses to wildfire. Using SL to explain variation in a natively chaotic environmental system is mechanism independent. Likely, the approach that we describe for combining SL with microbiome and biogeochemical parameters has practical applications across a range of issues in the environmental sciences where predicting responses would be useful.

Potential role for microbial ureolysis in the rapid formation of carbonate tufa mounds.

Modern carbonate tufa towers in the alkaline (~pH 9.5) Big Soda Lake (BSL), Nevada, exhibit rapid precipitation rates (exceeding 3 cm/year) and host diverse microbial communities. Geochemical indicators reveal that carbonate precipitation is, in part, promoted by the mixing of calcium-rich groundwater and carbonate-rich lake water, such that a microbial role for carbonate precipitation is unknown. Here, we characterize the BSL microbial communities and evaluate their potential effects on carbonate precipitation that may influence fast carbonate precipitation rates of the active tufa mounds of BSL. Small subunit rRNA gene surveys indicate a diverse microbial community living endolithically, in interior voids, and on tufa surfaces. Metagenomic DNA sequencing shows that genes associated with metabolisms that are capable of increasing carbonate saturation (e.g., photosynthesis, ureolysis, and bicarbonate transport) are abundant. Enzyme activity assays revealed that urease and carbonic anhydrase, two microbial enzymes that promote carbonate precipitation, are active in situ in BSL tufa biofilms, and urease also increased calcium carbonate precipitation rates in laboratory incubation analyses. We propose that, although BSL tufas form partially as a result of water mixing, tufa-inhabiting microbiota promote rapid carbonate authigenesis via ureolysis, and potentially via bicarbonate dehydration and CO2 outgassing by carbonic anhydrase. Microbially induced calcium carbonate precipitation in BSL tufas may generate signatures preserved in the carbonate microfabric, such as stromatolitic layers, which could serve as models for developing potential biosignatures on Earth and elsewhere.

Spatial and Temporal Constraints on the Composition of Microbial Communities in Subsurface Boreholes of the Edgar Experimental Mine.

The deep biosphere hosts uniquely adapted microorganisms overcoming geochemical extremes at significant depths within the crust of the Earth. Attention is required to understand the near subsurface and its continuity with surface systems, where numerous novel microbial members with unique physiological modifications remain to be identified. This surface-subsurface relationship raises key questions about networking of surface hydrology, geochemistry affecting near-subsurface microbial composition, and resiliency of subsurface ecosystems. Here, we apply molecular and geochemical approaches to determine temporal microbial composition and environmental conditions of filtered borehole fluid from the Edgar Experimental Mine (∼150 m below the surface) in Idaho Springs, CO. Samples were collected over a 4-year collection period from expandable packers deployed to accumulate fluid in previously drilled boreholes located centimeters to meters apart, revealing temporal evolution of borehole microbiology. Meteoric groundwater feeding boreholes demonstrated variable recharge rates likely due to a complex and undefined fracture system within the host rock. 16S rRNA gene analysis determined that unique microbial communities occupy the four boreholes examined. Two boreholes yielded sequences revealing the presence of Desulfosporosinus, Candidatus Nitrotoga, and Chelatococcus associated with endemic subsurface communities. Two other boreholes presented sequences related to nonsubsurface-originating microbiota. High concentration of sulfate along with detected sulfur reducing and oxidizing microorganisms suggests that sulfur related metabolic strategies are prominent within these near-subsurface boreholes. Overall, results indicate that microbial community composition in the near-subsurface is highly dynamic at very fine spatial scales (<20 cm) within fluid-rock equilibrated boreholes, which additionally supports the role of a relationship for surface geochemical processes infiltrating and influencing subsurface environments. IMPORTANCE The Edgar Experimental Mine, Idaho Springs, CO, provides inexpensive and open access to borehole investigations for subsurface microbiology studies. Understanding how microbial processes in the near subsurface are connected to surface hydrological influences is lacking. Investigating microbial communities of subsurface mine boreholes provides evidence of how geochemical processes are linked to biogeochemical processes within each borehole and the geochemical connectedness and mobility of surface influences. This study details microbial community composition and fluid geochemistry over spatial and temporal scales from boreholes within the Edgar Mine. These findings are relevant to biogeochemistry of near-surface mines, caves, and other voids across planetary terrestrial systems. In addition, this work can lead to understanding how microbial communities relate to both fluid-rock equilibration, and geochemical influences may enhance our understanding of subsurface molecular biological tools that aid mining economic practices to reflect biological signals for lucrative veins in the near subsurface.

Incident bone fracture and mortality in a large HIV cohort outpatient study, 2000-2017, USA.

We evaluated the association of bone fracture with mortality among persons with HIV, controlling for sociodemographic, behavioral, and clinical factors. Incident fracture was associated with 48% greater risk of all-cause mortality, underscoring the need for bone mineral density screening and fracture prevention. PURPOSE/INTRODUCTION: Low bone mineral density (BMD) and fracture are more common among persons with HIV (PWH) than those without HIV infection. We evaluated the association of bone fracture with mortality among PWH, controlling for sociodemographic, behavioral, and clinical factors. METHODS: We analyzed data from HIV Outpatient Study (HOPS) participants seen at nine US HIV clinics during January 1, 2000, through September 30, 2017. Incident fracture rates and post-fracture mortality were compared across four calendar periods. Cox proportional hazards analyses determined factors associated with all-cause mortality among all participants and those with incident fracture. RESULTS: Among 6763 HOPS participants, 504 (7.5%) had incident fracture (median age = 47 years) and 719 (10.6%) died. Of fractures, 135 (26.8%) were major osteoporotic (hip/pelvis, wrist, spine, arm/shoulder). During observation, 27 participants with major osteoporotic fractures died (crude mortality 2.97/100 person-years [PY]), and 48 with other site fractures died (crude mortality 2.51/100 PY). Post-fracture, age- and sex-adjusted all-cause mortality rates per 100 PY decreased from 8.5 during 2000-2004 to 1.9 during 2013-2017 (P<0.001 for trend). In multivariable analysis, incident fracture was significantly associated with all-cause mortality (Hazard Ratio 1.48, 95% confidence interval 1.15-1.91). Among 504 participants followed post-fracture, pulmonary, kidney, and cardiovascular disease, hepatitis C virus co-infection, and non-AIDS cancer, remained independently associated with all-cause mortality. CONCLUSIONS: Incident fracture was associated with 48% greater risk of all-cause mortality among US PWH in care, underscoring the need for BMD screening and fracture prevention. Although fracture rates among PWH increased during follow-up, post-fracture death rates decreased, likely reflecting advances in HIV care.

Genomic Analysis of the Yet-Uncultured Binatota Reveals Broad Methylotrophic, Alkane-Degradation, and Pigment Production Capacities.

The recent leveraging of genome-resolved metagenomics has generated an enormous number of genomes from novel uncultured microbial lineages yet left many clades undescribed. Here, we present a global analysis of genomes belonging to Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. All orders in Binatota encoded the capacity for aerobic methylotrophy using methanol, methylamine, sulfomethanes, and chloromethanes as the substrates. Methylotrophy in Binatota was characterized by order-specific substrate degradation preferences, as well as extensive metabolic versatility, i.e., the utilization of diverse sets of genes, pathways, and combinations to achieve a specific metabolic goal. The genomes also encoded multiple alkane hydroxylases and monooxygenases, potentially enabling growth on a wide range of alkanes and fatty acids. Pigmentation is inferred from a complete pathway for carotenoids (lycopene, ß- and γ-carotenes, xanthins, chlorobactenes, and spheroidenes) production. Further, the majority of genes involved in bacteriochlorophyll a, c, and d biosynthesis were identified, although absence of key genes and failure to identify a photosynthetic reaction center preclude proposing phototrophic capacities. Analysis of 16S rRNA databases showed the preferences of Binatota to terrestrial and freshwater ecosystems, hydrocarbon-rich habitats, and sponges, supporting their potential role in mitigating methanol and methane emissions, breakdown of alkanes, and their association with sponges. Our results expand the lists of methylotrophic, aerobic alkane-degrading, and pigment-producing lineages. We also highlight the consistent encountering of incomplete biosynthetic pathways in microbial genomes, a phenomenon necessitating careful assessment when assigning putative functions based on a set-threshold of pathway completion.IMPORTANCE A wide range of microbial lineages remain uncultured, yet little is known regarding their metabolic capacities, physiological preferences, and ecological roles in various ecosystems. We conducted a thorough comparative genomic analysis of 108 genomes belonging to the Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. We present evidence that members of the order Binatota specialize in methylotrophy and identify an extensive repertoire of genes and pathways mediating the oxidation of multiple one-carbon (C1) compounds in Binatota genomes. The occurrence of multiple alkane hydroxylases and monooxygenases in these genomes was also identified, potentially enabling growth on a wide range of alkanes and fatty acids. Pigmentation is inferred from a complete pathway for carotenoids production. We also report on the presence of incomplete chlorophyll biosynthetic pathways in all genomes and propose several evolutionary-grounded scenarios that could explain such a pattern. Assessment of the ecological distribution patterns of the Binatota indicates preference of its members to terrestrial and freshwater ecosystems characterized by high methane and methanol emissions, as well as multiple hydrocarbon-rich habitats and marine sponges.

Builders, tenants, and squatters: the origins of genetic material in modern stromatolites.

Micro-organisms have long been implicated in the construction of stromatolites. Yet, establishing a microbial role in modern stromatolite growth via molecular analysis is not always straightforward because DNA in stromatolites can have multiple origins. For example, the genomic material could represent the microbes responsible for the construction of the stromatolite (i.e., "builders"), microbes that inhabited the structure after it was built (i.e., "tenants"), or microbes/organic matter that were passively incorporated after construction from the water column or later diagenetic fluids (i.e., "squatters"). Disentangling the role of micro-organisms in stromatolite construction, already difficult in modern systems, becomes more difficult as organic signatures degrade, and their context is obscured. To evaluate our ability to accurately decipher the role of micro-organisms in stromatolite formation in geologically recent settings, 16/18S SSU rRNA gene sequences were analyzed from three systems where the context of growth was well understood: (a) an actively growing stromatolite from a silicic hot spring in Yellowstone National Park, Wyoming, where the construction of the structure is controlled by cyanobacteria; (b) a mixed carbonate and silica precipitate from Little Hot Creek, a hot spring in the Long Valley Caldera of California that has both abiogenic and biogenic components to accretion; and (c) a near-modern lacustrine carbonate stromatolite from Walker Lake, Nevada that is likely abiogenic. In all cases, the largest percentage of recovered DNA sequences, especially when focused on the deeper portions of the structures, belonged to either the tenant or squatter communities, not the actual builders. Once removed from their environmental context, correct interpretation of biology's role in stromatolite morphogenesis was difficult. Because high-throughput genomic analysis may easily lead to incorrect assumptions even in these modern and near-modern structures, caution must be exercised when interpreting micro-organismal involvement in the construction of accretionary structures throughout the rock record.

Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation.

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.

Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman.

Serpentinization can generate highly reduced fluids replete with hydrogen (H2) and methane (CH4), potent reductants capable of driving microbial methanogenesis and methanotrophy, respectively. However, CH4 in serpentinized waters is thought to be primarily abiogenic, raising key questions about the relative importance of methanogens and methanotrophs in the production and consumption of CH4 in these systems. Herein, we apply molecular approaches to examine the functional capability and activity of microbial CH4 cycling in serpentinization-impacted subsurface waters intersecting multiple rock and water types within the Samail Ophiolite of Oman. Abundant 16S rRNA genes and transcripts affiliated with the methanogenic genus Methanobacterium were recovered from the most alkaline (pH, >10), H2- and CH4-rich subsurface waters. Additionally, 16S rRNA genes and transcripts associated with the aerobic methanotrophic genus Methylococcus were detected in wells that spanned varied fluid geochemistry. Metagenomic sequencing yielded genes encoding homologs of proteins involved in the hydrogenotrophic pathway of microbial CH4 production and in microbial CH4 oxidation. Transcripts of several key genes encoding methanogenesis/methanotrophy enzymes were identified, predominantly in communities from the most hyperalkaline waters. These results indicate active methanogenic and methanotrophic populations in waters with hyperalkaline pH in the Samail Ophiolite, thereby supporting a role for biological CH4 cycling in aquifers that undergo low-temperature serpentinization.IMPORTANCE Serpentinization of ultramafic rock can generate conditions favorable for microbial methane (CH4) cycling, including the abiotic production of hydrogen (H2) and possibly CH4 Systems of low-temperature serpentinization are geobiological targets due to their potential to harbor microbial life and ubiquity throughout Earth's history. Biomass in fracture waters collected from the Samail Ophiolite of Oman, a system undergoing modern serpentinization, yielded DNA and RNA signatures indicative of active microbial methanogenesis and methanotrophy. Intriguingly, transcripts for proteins involved in methanogenesis were most abundant in the most highly reacted waters that have hyperalkaline pH and elevated concentrations of H2 and CH4 These findings suggest active biological methane cycling in serpentinite-hosted aquifers, even under extreme conditions of high pH and carbon limitation. These observations underscore the potential for microbial activity to influence the isotopic composition of CH4 in these systems, which is information that could help in identifying biosignatures of microbial activity on other planets.

Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

Microbial Metabolic Redundancy Is a Key Mechanism in a Sulfur-Rich Glacial Ecosystem.

Biological sulfur cycling in polar, low-temperature ecosystems is an understudied phenomenon in part due to difficulty of access and the dynamic nature of glacial environments. One such environment where sulfur cycling is known to play an important role in microbial metabolisms is located at Borup Fiord Pass (BFP) in the Canadian High Arctic. Here, transient springs emerge from ice near the terminus of a glacier, creating a large area of proglacial aufeis (spring-derived ice) that is often covered in bright yellow/white sulfur, sulfate, and carbonate mineral precipitates accompanied by a strong odor of hydrogen sulfide. Metagenomic sequencing of samples from multiple sites and of various sample types across the BFP glacial system produced 31 metagenome-assembled genomes (MAGs) that were queried for sulfur, nitrogen, and carbon cycling/metabolism genes. An abundance of sulfur cycling genes was widespread across the isolated MAGs and sample metagenomes taxonomically associated with the bacterial classes Alphaproteobacteria and Gammaproteobacteria and Campylobacteria (formerly the Epsilonproteobacteria). This corroborates previous research from BFP implicating Campylobacteria as the primary class responsible for sulfur oxidation; however, data reported here suggested putative sulfur oxidation by organisms in both the alphaproteobacterial and gammaproteobacterial classes that was not predicted by previous work. These findings indicate that in low-temperature, sulfur-based environments, functional redundancy may be a key mechanism that microorganisms use to enable coexistence whenever energy is limited and/or focused by redox chemistry.IMPORTANCE A unique environment at Borup Fiord Pass is characterized by a sulfur-enriched glacial ecosystem in the low-temperature Canadian High Arctic. BFP represents one of the best terrestrial analog sites for studying icy, sulfur-rich worlds outside our own, such as Europa and Mars. The site also allows investigation of sulfur-based microbial metabolisms in cold environments here on Earth. Here, we report whole-genome sequencing data that suggest that sulfur cycling metabolisms at BFP are more widely used across bacterial taxa than predicted. From our analyses, the metabolic capability of sulfur oxidation among multiple community members appears likely due to functional redundancy present in their genomes. Functional redundancy, with respect to sulfur-oxidation at the BFP sulfur-ice environment, may indicate that this dynamic ecosystem hosts microorganisms that are able to use multiple sulfur electron donors alongside other metabolic pathways, including those for carbon and nitrogen.

Insights into the dynamics between viruses and their hosts in a hot spring microbial mat.

Our current knowledge of host-virus interactions in biofilms is limited to computational predictions based on laboratory experiments with a small number of cultured bacteria. However, natural biofilms are diverse and chiefly composed of uncultured bacteria and archaea with no viral infection patterns and lifestyle predictions described to date. Herein, we predict the first DNA sequence-based host-virus interactions in a natural biofilm. Using single-cell genomics and metagenomics applied to a hot spring mat of the Cone Pool in Mono County, California, we provide insights into virus-host range, lifestyle and distribution across different mat layers. Thirty-four out of 130 single cells contained at least one viral contig (26%), which, together with the metagenome-assembled genomes, resulted in detection of 59 viruses linked to 34 host species. Analysis of single-cell amplification kinetics revealed a lack of active viral replication on the single-cell level. These findings were further supported by mapping metagenomic reads from different mat layers to the obtained host-virus pairs, which indicated a low copy number of viral genomes compared to their hosts. Lastly, the metagenomic data revealed high layer specificity of viruses, suggesting limited diffusion to other mat layers. Taken together, these observations indicate that in low mobility environments with high microbial abundance, lysogeny is the predominant viral lifestyle, in line with the previously proposed "Piggyback-the-Winner" theory.