Parapatric speciation of Meiothermus in serpentinite-hosted aquifers in Oman.

The factors that control the distribution and evolution of microbial life in subsurface environments remain enigmatic due to challenges associated with sampling fluids from discrete depth intervals via boreholes while avoiding mixing of fluids. Here, using an inflatable packer system, fracture waters were isolated and collected from three discrete depth intervals spanning >130 m in a borehole intersecting an ultramafic rock formation undergoing serpentinization in the Samail Ophiolite, Sultanate of Oman. Near surface aquifer waters were moderately reducing and had alkaline pH while deeper aquifer waters were reduced and had hyperalkaline pH, indicating extensive influence by serpentinization. Metagenomic sequencing and analysis of DNA from filtered biomass collected from discrete depth intervals revealed an abundance of aerobes in near surface waters and a greater proportion of anaerobes at depth. Yet the abundance of the putatively obligate aerobe, Meiothermus, increased with depth, providing an opportunity to evaluate the influence of chemical and spatial variation on its distribution and speciation. Two clades of Meiothermus metagenome assembled genomes (MAGs) were identified that correspond to surface and deep populations termed Types I (S) and II (D), respectively; both clades comprised an apparently Oman-specific lineage indicating a common ancestor. Type II (D) clade MAGs encoded fewer genes and were undergoing slower genome replication as inferred from read mapping. Further, single nucleotide variants (SNVs) and mobile genetic elements identified among MAGs revealed detectable, albeit limited, evidence for gene flow/recombination between spatially segregated Type I (S) and Type II (D) populations. Together, these observations indicate that chemical variation generated by serpentinization, combined with physical barriers that reduce/limit dispersal and gene flow, allowed for the parapatric speciation of Meiothermus in the Samail Ophiolite or a geologic precursor. Further, Meiothermus genomic data suggest that deep and shallow aquifer fluids in the Samail Ophiolite may mix over shorter time scales than has been previously estimated from geochemical data.

ATP Is a Major Determinant of Phototrophic Bacterial Longevity in Growth Arrest.

How bacteria transition into growth arrest as part of stationary phase has been well-studied, but our knowledge of features that help cells to stay alive in the following days and weeks is incomplete. Most studies have used heterotrophic bacteria that are growth-arrested by depletion of substrates used for both biosynthesis and energy generation, making is difficult to disentangle the effects of the two. In contrast, when grown anaerobically in light, the phototrophic bacterium Rhodopseudomonas palustris generates ATP from light via cyclic photophosphorylation, and builds biomolecules from organic substrates, such as acetate. As such, energy generation and carbon utilization are independent from one another. Here, we compared the physiological and molecular responses of R. palustris to growth arrest caused by carbon source depletion in light (energy-replete) and dark (energy-depleted) conditions. Both sets of cells remained viable for 6 to 10 days, at which point dark-incubated cells lost viability, whereas light-incubated cells remained fully viable for 60 days. Dark-incubated cells were depleted in intracellular ATP prior to losing viability, suggesting that ATP depletion is a cause of cell death. Dark-incubated cells also shut down measurable protein synthesis, whereas light-incubated cells continued to synthesize proteins at low levels. Cells incubated in both conditions continued to transcribe genes. We suggest that R. palustris may completely shut down protein synthesis in dark, energy-depleted, conditions as a strategy to survive the nighttime hours of day/night cycles it experiences in nature, where there is a predictable source of energy in the form of sunlight only during the day. IMPORTANCE The molecular and physiological basis of bacterial longevity in growth arrest is important to investigate for several reasons. Such investigations could improve treatment of chronic infections, advance use of non-growing bacteria as biocatalysts to make high yields of value-added products, and improve estimates of microbial activities in natural habitats, where cells are often growing slowly or not at all. Here, we compared survival of the phototrophic bacterium Rhodopseudomonas palustris under conditions where it generates ATP (incubation in light), and where it does not generate ATP (incubation in dark) to directly assess effects of energy depletion on long-term viability. We found that ATP is important for long-term survival over weeks. However, R. palustris survives 12 h periods of ATP depletion without loss of viability, apparently in anticipation of sunrise and restoration of its ability to generate ATP. Our work suggests that cells respond to ATP depletion by shutting down protein synthesis.

Transformation of low-molecular-weight organic acids by microbial endoliths in subsurface mafic and ultramafic igneous rock.

A growing body of work indicates that continental subsurface rocks host a substantial portion of the Earth's biosphere. However, the activities of microbial cells inhabiting pore spaces and microfractures in subsurface rocks remain underexplored. Here, we develop and optimize microcosm assays to detect organic acid transformation activities of cells residing in mafic to ultramafic igneous rocks. Application of this assay to gabbro core from the Stillwater Mine, Montana, USA, revealed maximal methane production from acetate at temperatures approximating that of the mine. Controls show that these activities are not due to contamination introduced during drilling, exhumation, or laboratory processing of the core. The assay was then applied to rocks cored from the Samail Ophiolite, Oman, which is undergoing low-temperature serpentinization. Production of (i) carbon dioxide from acetate and formate and (ii) methane from formate were detected in a dunite/harzburgite rock core interfacing pH 9.6 waters, and estimates of microbial activities were up to three orders of magnitude higher in the rock core pore space than in corresponding waters. The detection of endolithic microbial activities in igneous rocks has implications for life detection on other planetary bodies where similar rock types prevail, such as Mars, Europa and Enceladus.

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.

Geologic legacy spanning >90 years explains unique Yellowstone hot spring geochemistry and biodiversity.

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

Physiological adaptations to serpentinization in the Samail Ophiolite, Oman.

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.

Origin and Evolution of Flavin-Based Electron Bifurcating Enzymes.

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.

Staphylococcus aureus SaeR/S-Regulated Factors Decrease Monocyte-Derived Tumor Necrosis Factor-α to Reduce Neutrophil Bactericidal Activity.

Background: The ability of Staphylococcus aureus to evade killing by human neutrophils significantly contributes to disease progression. In this study, we characterize an influential role for the S. aureus SaeR/S 2-component gene regulatory system in suppressing monocyte production of tumor necrosis factor alpha (TNF-α) to subsequently influence human neutrophil priming. Methods: Using flow cytometry and TNF-α specific enzyme-linked immunosorbent assays we identify the primary cellular source of TNF-α in human blood and in purified peripheral blood mononuclear cells (PBMCs) during interaction with USA300 and an isogenic saeR/S deletion mutant (USA300∆saeR/S). Assays with conditioned media from USA300 and USA300∆saeR/S exposed PBMCs were used to investigate priming on neutrophil bactericidal activity. Results: TNF-α production from monocytes was significantly reduced following challenge with USA300 compared to USA300∆saeR/S. We observed that priming of neutrophils using conditioned medium from peripheral blood mononuclear cells stimulated with USA300∆saeR/S significantly increased neutrophil bactericidal activity against USA300 relative to unprimed neutrophils and neutrophils primed with USA300 conditioned medium. The increased neutrophil bactericidal activity was associated with enhanced reactive oxygen species production that was significantly influenced by elevated TNF-α concentrations. Conclusions: Our findings identify an immune evasion strategy used by S. aureus to impede neutrophil priming and subsequent bactericidal activity.