The formation and fate of pyrite (FeS2) modulates global iron, sulfur, carbon, and oxygen biogeochemical cycles and has done so since early in Earth’s geological history. A longstanding paradigm is that FeS2 is stable at low temperature and is unavailable to microorganisms in the absence of oxygen and oxidative weathering. Here, we show that methanogens can catalyze the reductive dissolution of FeS2 at low temperature (≤38 °C) and utilize dissolution products to meet cellular iron and sulfur demands associated with the biosynthesis of simple and complex co-factors. Direct access to FeS2 is required to catalyze its reduction and/or to assimilate iron monosulfide that likely forms through coupled reductive dissolution and precipitation, consistent with close associations observed between cells and FeS2. These findings demonstrate that FeS2 is bioavailable to anaerobic methanogens and can be mobilized in low temperature anoxic environments. Given that methanogens evolved at least 3.46 Gya, these data indicate that the microbial contribution to the iron and sulfur cycles in ancient and contemporary anoxic environments may be more complex and robust than previously recognized, with impacts on the sources and sinks of iron and sulfur and other bio-essential and thiophilic elements such as nickel and cobalt.

Methanogens have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, deploy, and store these elements and how this, in turn, affects their physiology. Methanogens were recently shown to reduce pyrite (FeS 2 ) generating aqueous iron-sulfide (FeS (aq) ) clusters that are likely assimilated as a source of Fe and S. Here, we compare the phenotype of Methanococcus voltae when grown with FeS 2 or ferrous iron (Fe(II)) and sulfide (HS - ). FeS 2 -grown cells are 33% smaller yet have 193% more Fe than Fe(II)/HS - -grown cells. Whole cell EPR revealed similar distributions of paramagnetic Fe, although FeS 2 -grown cells showed a broad spectral feature attributed to intracellular thioferrate-like nanoparticles. Differential proteomic analyses showed similar expression of core methanogenesis enzymes, indicating that Fe and S source does not substantively alter the energy metabolism of cells. However, a homolog of the Fe(II) transporter FeoB and its putative transcriptional regulator DtxR were up-expressed in FeS 2 -grown cells, suggesting that cells sense Fe(II) limitation. Two homologs of IssA, a protein putatively involved in coordinating thioferrate nanoparticles, were also up-expressed in FeS 2 -grown cells. We interpret these data to indicate that, in FeS 2 -grown cells, DtxR cannot sense Fe(II) and therefore cannot down-regulate FeoB. We suggest this is due to the transport of Fe(II) complexed with sulfide (FeS (aq) ) leading to excess Fe that is sequestered by IssA as a thioferrate-like species. This model provides a framework for the design of targeted experiments aimed at further characterizing Fe acquisition and homeostasis in M. voltae and other methanogens. IMPORTANCE FeS 2 is the most abundant sulfide mineral in the Earth’s crust and is common in environments inhabited by methanogenic archaea. FeS 2 can be reduced by methanogens, yielding aqueous FeS (aq) clusters that are thought to be a source of Fe and S. Here, we show that growth of Methanococcus voltae on FeS 2 results in smaller cell size and higher Fe content per cell, with Fe likely stored intracellularly as thioferrate-like nanoparticles. Fe(II) transporters and storage proteins were up-regulated in FeS 2 -grown cells. These responses are interpreted to result from cells incorrectly sensing Fe(II) limitation due to assimilation of Fe(II) as FeS (aq) . These findings have implications for our understanding of how Fe/S availability influences methanogen physiology and the biogeochemical cycling of these elements.

Pyrite (FeS2) has a very low solubility and therefore has historically been considered a sink for iron (Fe) and sulfur (S) and unavailable to biology in the absence of oxygen and oxidative weathering. Anaerobic methanogens were recently shown to reduce FeS2 and assimilate Fe and S reduction products to meet nutrient demands. However, the mechanism of FeS2 mineral reduction and the forms of Fe and S assimilated by methanogens remained unclear. Thermodynamic calculations described herein indicate that H2 at aqueous concentrations as low as 10–10 M favors the reduction of FeS2, with sulfide (HS–) and pyrrhotite (Fe1–xS) as products; abiotic laboratory experiments confirmed the reduction of FeS2 with dissolved H2 concentrations greater than 1.98 × 10–4 M H2. Growth studies of Methanosarcina barkeri provided with FeS2 as the sole source of Fe and S resulted in H2 production but at concentrations too low to drive abiotic FeS2 reduction, based on abiotic laboratory experimental data. A strain of M. barkeri with deletions in all [NiFe]-hydrogenases maintained the ability to reduce FeS2 during growth, providing further evidence that extracellular electron transport (EET) to FeS2 does not involve H2 or [NiFe]-hydrogenases. Physical contact between cells and FeS2 was required for mineral reduction but was not required to obtain Fe and S from dissolution products. The addition of a synthetic electron shuttle, anthraquinone-2,6-disulfonate, allowed for biological reduction of FeS2 when physical contact between cells and FeS2 was prohibited, indicating that exogenous electron shuttles can mediate FeS2 reduction. Transcriptomics experiments revealed upregulation of several cytoplasmic oxidoreductases during growth of M. barkeri on FeS2, which may indicate involvement in provisioning low potential electrons for EET to FeS2. Collectively, the data presented herein indicate that reduction of insoluble FeS2 by M. barkeri occurred via electron transfer from the cell surface to the mineral surface resulting in the generation of soluble HS– and mineral-associated Fe1–xS. Solubilized Fe(II), but not HS–, from mineral-associated Fe1–xS reacts with aqueous HS– yielding aqueous iron sulfur clusters (FeSaq) that likely serve as the Fe and S source for methanogen growth and activity. FeSaq nucleation and subsequent precipitation on the surface of cells may result in accelerated EET to FeS2, resulting in positive feedback between cell activity and FeS2 reduction.

Summary 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.

Microbial mat communities are associated with extensive (∼700 km2) and morphologically variable carbonate structures, termed microbialites, in the hypersaline Great Salt Lake (GSL), Utah. However, whether the composition of GSL mat communities covaries with microbialite morphology and lake environment is unknown. Moreover, the potential adaptations that allow the establishment of these extensive mat communities at high salinity (14% to 17% total salts) are poorly understood. To address these questions, microbial mats were sampled from seven locations in the south arm of GSL representing different lake environments and microbialite morphologies. Despite the morphological differences, microbialite-associated mats were taxonomically similar and were dominated by the cyanobacterium Euhalothece and several heterotrophic bacteria. Metagenomic sequencing of a representative mat revealed Euhalothece and subdominant Thiohalocapsa populations that harbor the Calvin cycle and nitrogenase, suggesting they supply fixed carbon and nitrogen to heterotrophic bacteria. Fifteen of the next sixteen most abundant taxa are inferred to be aerobic heterotrophs and, surprisingly, harbor reaction center, rhodopsin, and/or bacteriochlorophyll biosynthesis proteins, suggesting aerobic photoheterotrophic (APH) capabilities. Importantly, proteins involved in APH are enriched in the GSL community relative to that in microbialite mat communities from lower salinity environments. These findings indicate that the ability to integrate light into energy metabolism is a key adaptation allowing for robust mat development in the hypersaline GSL. IMPORTANCE The earliest evidence of life on Earth is from organosedimentary structures, termed microbialites, preserved in 3.481-billion-year-old (Ga) rocks. Phototrophic microbial mats form in association with an ∼700-km2 expanse of morphologically diverse microbialites in the hypersaline Great Salt Lake (GSL), Utah. Here, we show taxonomically similar microbial mat communities are associated with morphologically diverse microbialites across the lake. Metagenomic sequencing reveals an abundance and diversity of autotrophic and heterotrophic taxa capable of harvesting light energy to drive metabolism. The unexpected abundance of and diversity in the mechanisms of harvesting light energy observed in GSL mat populations likely function to minimize niche overlap among coinhabiting taxa, provide a mechanism(s) to increase energy yield and osmotic balance during salt stress, and enhance fitness. Together, these physiological benefits promote the formation of robust mats that, in turn, influence the formation of morphologically diverse microbialite structures that can be imprinted in the rock record.