Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils

The possibility thatMethanothrix (formerlyMethanosaeta ) andGeobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) ofGeobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated withGeobacter species, but sequences most closely related to putative e-pilin genes from genera such asDesulfobacterium ,Deferribacter ,Geoalkalibacter , andDesulfobacula , were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered withGeobacter species, and the finding thatGeobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribedGeobacter genes. In addition, homologs of genes encoding OmcS, ac -type cytochrome associated with the e-pili ofG. sulfurreducens and required for DIET, were also highly expressed in the soils.Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction inMethanothrix . Thus, these results are consistent with a model in whichGeobacter species were providing electrons toMethanothrix species for methane production through electrical connections of e-pili.IMPORTANCE Methanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons forMethanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution thatMethanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that whenMethanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus,Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason thatGeobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation withMethanothrix spp., and possibly other methanogens, via DIET.