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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 contribute 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. 

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. 

ABSTRACT 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, β- 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 (C 1 ) 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. 

Abstract 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. 

Abstract 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.