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Nickel (Ni) is a key component of the active site metallocofactors of numerous enzymes required for methanogenesis, including [NiFe]-hydrogenase, carbon monoxide dehydrogenase, and methyl CoM reductase, leading to a high demand for Ni among methanogens. However, methanogens often inhabit euxinic environments that favor the sequestration of nickel as metal-sulfide minerals, such as nickelian pyrite [(Ni, Fe)S2], that have low solubilities and that are not considered bioavailable. Recently, however, several different model methanogens (Methanosarcina barkeri, Methanococcus voltae, Methanococcus maripaludis) were shown to reductively dissolve pyrite (FeS2) and to utilize dissolution products to meet iron and sulfur biosynthetic demands. Here, using M. barkeri Fusaro, and laboratory-synthesized (Ni, Fe)S2 that was physically isolated from cells using dialysis membranes, we show that trace nickel (<20 nM) abiotically solubilized from the mineral can support methanogenesis and limited growth, roughly fivefold less than the minimum concentration known to support methanogenesis. Furthermore, when provided direct contact with (Ni, Fe)S2, M. barkeri promoted the reductive dissolution of (Ni, Fe)S2 and assimilated solubilized nickel, iron, and sulfur as its sole source of these elements. Cells that reductively dissolved (Ni, Fe)S2 bioaccumulated approximately fourfold more nickel than those grown with soluble nickel and sulfide but had similar metabolic coupling efficiencies. While the mechanism for Ni uptake in archaeal methanogens is not known, homologs of the bacterial Nik uptake system were shown to be ubiquitous across methanogen genomes. Collectively, these observations indicate that (Ni, Fe)S2 is bioavailable in anoxic environments and that methanogens can convert this mineral into nickel-, iron-, and sulfur-containing metalloenzymes to support methanogenesis and growth. [ABSTRACT FROM AUTHOR]

Pyrite (FeS 2) is the most abundant iron sulfide mineral in Earth's crust. Until recently, FeS 2 has been considered a sink for iron (Fe) and sulfur (S) at low temperature in the absence of oxygen or oxidative weathering, making these elements unavailable to biology. However, anaerobic methanogens can transfer electrons extracellularly to reduce FeS 2 via direct contact with the mineral. Reduction of FeS 2 occurs through a multistep process that generates aqueous sulfide (HS–) and FeS 2 -associated pyrrhotite (Fe 1-x S). Subsequent dissolution of Fe 1-x S provides Fe(II) (aq) , but not HS–, that rapidly complexes with HS– (aq) generated from FeS 2 reduction to form soluble iron sulfur clusters [ n FeS (aq) ]. Cells assimilate n FeS (aq) to meet Fe/S nutritional demands by mobilizing and hyperaccumulating Fe and S from FeS 2. As such, reductive dissolution of FeS 2 by methanogens has important implications for element cycling in anoxic habitats, both today and in the geologic past. Methanogens can reductively dissolve pyrite (FeS 2) and mobilize iron (Fe) and sulfur (S) under anaerobic conditions. Biological reduction of FeS 2 takes place via direct extracellular electron transfer (EET) that likely involves electrically conductive components of the S-layer or extracellular polymeric matrix. Fe(II) solubilized from Fe 1-x S that precipitates on the surface of FeS 2 during reduction combines with HS– released during FeS 2 reduction to form n FeS (aq) clusters that are likely assimilated to meet Fe and S nutritional demands. Growth with FeS 2 causes cells to perceive Fe limitation due to transport of n FeS (aq) rather than ionic Fe(II), thereby leading to hyperaccumulation of Fe in cells as putative thioferrate-like particles. Reduction of FeS 2 , assimilation of n FeS (aq) , and transformation into functional biocatalysts in methanogens provides a potential path to explore the transition from abiotic, mineral-based catalysis to biocatalysis on early Earth. [ABSTRACT FROM AUTHOR]

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– x S) 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– x S. Solubilized Fe(II), but not HS–, from mineral-associated Fe1– x S reacts with aqueous HS– yielding aqueous iron sulfur clusters (FeS aq ) that likely serve as the Fe and S source for methanogen growth and activity. FeS aq 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. [ABSTRACT FROM AUTHOR]