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.

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.

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Twelve evolutionarily unrelated oxidoreductases form enzyme complexes that catalyze the simultaneous coupling of exergonic and endergonic oxidation–reduction reactions to circumvent thermodynamic barriers and minimize free energy loss in a process known as flavin-based electron bifurcation. Common to these 12 bifurcating (Bf) enzymes are protein-bound flavin, the proposed site of bifurcation, and the electron carrier ferredoxin. Despite the documented role of Bf enzymes in balancing the redox state of intracellular electron carriers and in improving the efficiency of cellular metabolism, a comprehensive description of the diversity and evolutionary history of Bf enzymes is lacking. Here, we report the taxonomic distribution, functional diversity, and evolutionary history of Bf enzyme homologs in 4,588 archaeal, bacterial, and eukaryal genomes and 3,136 community metagenomes. Bf homologs were primarily detected in the genomes of anaerobes, including those of sulfate-reducers, acetogens, fermenters, and methanogens. Phylogenetic analyses of Bf enzyme catalytic subunits (oxidoreductases) suggest they were not a property of the Last Universal Common Ancestor of Archaea and Bacteria, which is consistent with the limited and unique taxonomic distributions of enzyme homologs among genomes. Further, phylogenetic analyses of oxidoreductase subunits reveal that non-Bf homologs predate Bf homologs. These observations indicate that multiple independent recruitments of flavoproteins to existing oxidoreductases enabled coupling of numerous new electron Bf reactions. Consistent with the role of these enzymes in the energy metabolism of anaerobes, homologs of Bf enzymes were enriched in metagenomes from subsurface environments relative to those from surface environments. Phylogenetic analyses of homologs from metagenomes reveal that the earliest evolving homologs of most Bf enzymes are from subsurface environments, including fluids from subsurface rock fractures and hydrothermal systems. Collectively, these data suggest strong selective pressures drove the emergence of Bf enzyme complexes via recruitment of flavoproteins that allowed for an increase in the efficiency of cellular metabolism and improvement in energy capture in anaerobes inhabiting a variety of subsurface anoxic habitats where the energy yield of oxidation-reduction reactions is generally low.