Mechanisms for Electron Uptake byMethanosarcina acetivoransDuring Direct Interspecies Electron Transfer

Abstract

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in co-culture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in co-culture versus pure culture growth on acetate revealed that transcripts for the outer-surface, multi-heme, c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based co-culture as well as the archaella-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based co-cultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen.

Different Routes for Direct Interspecies Electron Transfer with Diverse Electron-Accepting Partners

“…Gene expression patterns suggest that the electron-accepting partner influences routes for DIET DIET between G. metallireducens and G. sulfurreducens was compared to DIET between G. metallireducens and three Methanosarcina species: M. barkeri (MB), a Type I Methanosarcina that lacks outer-surface cytochromes (15) and M. acetivorans (MA), and M. subterranea (MS), Type II Methanosarcina with the outer-surface multi-heme ctype cytochrome MmcA that serves an essential electrical contact for DIET (9).…”

“…Constructing Geobacter strains that express poorly conductive pili is a method for evaluating the evaluating the importance of pili conductivity in extracellular electron exchange without disrupting the proper expression of outer surface cytochromes that also play a key role in DIET (36)(37)(38)(39)(40). A strain of G. metallireducens that expresses poorly conductive pili was previously found to be defective in establishing DIET co-cultures with G. sulfurreducens (41) and the Type II Methanosarcina species, M. acetivorans (9). A co-culture initiated with G. metallireducens expressing poorly conductive pili and the Type I Methanosarcina species, M. barkeri, was delayed in converting ethanol to methane, but adapted to grow in co-culture at rates that were similar to the wild-type co-cultures after four transfers (Figure 6).…”

“…Until now, studies on the routes for interspecies electron transfer during DIET have primarily focused on the mechanisms of electron uptake by the electron-accepting partner (5)(6)(7)(8)(9). These studies have demonstrated that the electrical connects on the outer cell surface of electron-accepting partners can be dramatically different.…”

“…The electrical contacts for DIET in these methanogens are unknown. However, another methanogen, Methanosarcina acetivorans, expresses an outer-surface multi-heme c-type cytochrome, MmcA, that is a key electrical contact for DIET (9). This major difference in mechanisms for DIET-based electron uptake between M. barkeri and M. acetivorans is just one of the physiological dissimilarities between two clades of Methanosarcina species.…”

“…The electrical contacts for DIET in these methanogens are unknown. However, another methanogen, Methanosarcina acetivorans , expresses an outer-surface multi-heme c -type cytochrome, MmcA, that is a key electrical contact for DIET (9).…”