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Item Alternative sources of molybdenum for Methanococcus maripaludis and their implication for the evolution of molybdoenzymes(Springer Science and Business Media LLC, 2024-10) Payne, Devon; Keller, Lisa M.; Larson, James; Bothner, Brian; Colman, Daniel; Boyd, Eric S.Molybdoenzymes are essential in global nitrogen, carbon, and sulfur cycling. To date, the only known bioavailable source of molybdenum (Mo) is molybdate. However, in the sulfidic and anoxic (euxinic) habitats that predominate in modern subsurface environments and that were pervasive prior to Earth’s widespread oxygenation, Mo occurs as soluble tetrathiomolybdate ion and molybdenite mineral that is not known to be bioavailable. This presents a paradox for how organisms obtain Mo to support molybdoenzymes in these environments. Here, we show that tetrathiomolybdate and molybdenite sustain the high Mo demand of a model anaerobic methanogen, Methanococcus maripaludis, grown via Mo-dependent formate dehydrogenase, formylmethanofuran dehydrogenase, and nitrogenase. Cells grown with tetrathiomolybdate and molybdenite have similar growth kinetics, Mo content, and transcript levels of proteins involved in Mo transport and cofactor biosynthesis when compared to those grown with molybdate, implying similar mechanisms of transport and cofactor biosynthesis. These results help to reconcile the paradox of how Mo is acquired in modern and ancient anaerobes and provide new insight into how molybdoenzymes could have evolved prior to Earth’s oxygenation.Item Impact of mineral and non-mineral sources of iron and sulfur on the metalloproteome of Methanosarcina barkeri(American Society for Microbiology, 2024-07) Larson, James; Tokmina-Lukaszewska, Monika; Payne, Devon; Spietz, Rachel L.; Fausset, Hunter; Alam, Md Gahangir; Brekke, Brooklyn K.; Pauley, Jordan; Hasenoehrl, Ethan J.; Shepard, Eric M.; Boyd, Eric S.; Bothner, BrianMethanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS2). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS-) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS2 to sustain growth. As such, medium supplied with FeS2 should lead to higher availability of transition metals when compared to medium supplied with HS-. Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS- as sole S source) versus non-sulfidic (cells provided with FeS2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.Item Reductive dissolution of pyrite by methanogens and its physiological and ecological consequences(Montana State University - Bozeman, College of Agriculture, 2022) Payne, Devon; Chairperson, Graduate Committee: Eric Boyd; This is a manuscript style paper that includes co-authored chapters.All life requires iron and sulfur, in particular, for use in metallocofactors of enzymes that catalyze chemistry that is essential for metabolism. In anaerobic environments, iron and sulfur are typically found in their reduced forms (ferrous iron and sulfide, respectively) that will react and form insoluble iron-sulfide minerals, such as pyrite. A consequence of this is that either iron or sulfur are typically limiting in solution, raising the question as to how anaerobes acquire these essential elements under such conditions. Here, it is demonstrated that anaerobic methanogens can reduce pyrite to release iron and sulfur that are assimilated by the cells to meet biosynthetic demands. Through a combination of growth experiments, -omics analyses, and microscopy, a model for the reductive dissolution of pyrite was established. In this model, direct contact between cells and pyrite is required for mineral reduction. When in direct contact, pyrite is reduced and sulfide is released, leaving a pyrrhotite secondary mineral on the surface. Iron solubilized from pyrrhotite reacts with sulfide in the growth medium to yield aqueous iron sulfur clusters that are assimilated by cells. Cells grown on pyrite exhibit phenotypic differences in comparison to traditionally grown cells provided with ferrous iron and sulfide. At a morphological level, pyrite-grown cells were 33% smaller than traditionally grown cells and hyperaccumulated iron as an intracellular mineral. When grown under nitrogen-fixing conditions, cells grown on pyrite had higher cell densities, growth yields, and growth rates in comparison to traditionally grown cells. Molybdate transporters were down expressed in pyrite-grown, nitrogen-fixing cells relative to traditionally-grown cells, consistent with sulfide limiting molybdate availability in the latter condition. Moreover, pyrite-grown cells could fix nitrogen at ~100-fold lower molybdenum concentration than traditionally grown cells, indicating differences in molybdenum requirements based on the iron and sulfur source provided. Together, these data highlight that in contemporary anoxic environments, iron-sulfide minerals are an important and even preferred source of iron and sulfur for methanogens. These findings provide insight into how ancient methanogens could have acquired iron and sulfur on an anoxic early Earth when one or both of these elements were likely only available as metal sulfides.Item Proteomic Analysis of Methanococcus voltae Grown in the Presence of Mineral and Nonmineral Sources of Iron and Sulfur(American Society for Microbiology, 2022-08) Steward, Katherine F.; Payne, Devon; Kincannon, Will; Johnson, Christina; Lensing, Malachi; Fausset, Hunter; Németh, Brigitta; Shepard, Eric M.; Broderick, William E.; Broderick, Joan B.; Dubois, Jen; Bothner, BrianClusters of iron and sulfur are key components of the active sites of enzymes that facilitate microbial conversion of light or electrical energy into chemical bonds. The proteins responsible for transporting iron and sulfur into cells and assembling these elements into metal clusters are not well understood.Item Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction(Frontiers Media SA, 2022-05) Lange Spietz, Rachel K.; Payne, Devon; Kulkarni, Gargi; Metcalf, William W.; Roden, Eric E.; Boyd, Eric S.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.