Examining Pathways of Iron and Sulfur Acquisition, Trafficking, Deployment, and Storage in Mineral-Grown Methanogen Cells

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 compared the phenotypes of Methanococcus voltae 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 electron paramagnetic resonance 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 downregulate 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 upregulated 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.