Transcriptomic and genetic analysis of direct interspecies electron transfer

Abstract

The possibility that metatranscriptomic analysis could distinguish between direct interspecies electron transfer (DIET) and H2 interspecies transfer (HIT) in anaerobic communities was investigated by comparing gene transcript abundance in cocultures in which Geobacter sulfurreducens was the electron-accepting partner for either Geobacter metallireducens, which performs DIET, or Pelobacter carbinolicus, which relies on HIT. Transcript abundance for G. sulfurreducens uptake hydrogenase genes was 7-fold lower in cocultures with G. metallireducens than in cocultures with P. carbinolicus, consistent with DIET and HIT, respectively, in the two cocultures. Transcript abundance for the pilus-associated cytochrome OmcS, which is essential for DIET but not for HIT, was 240-fold higher in the cocultures with G. metallireducens than in cocultures with P. carbinolicus. The pilin gene pilA was moderately expressed despite a mutation that might be expected to repress pilA expression. Lower transcript abundance for G. sulfurreducens genes associated with acetate metabolism in the cocultures with P. carbinolicus was consistent with the repression of these genes by H2 during HIT. Genes for the biogenesis of pili and flagella and several c-type cytochrome genes were among the most highly expressed in G. metallireducens. Mutant strains that lacked the ability to produce pili, flagella, or the outer surface c-type cytochrome encoded by Gmet_2896 were not able to form cocultures with G. sulfurreducens. These results demonstrate that there are unique gene expression patterns that distinguish DIET from HIT and suggest that metatranscriptomics may be a promising route to investigate interspecies electron transfer pathways in more-complex environments.

Fig 1

Heat map comparison of expression levels of genes associated with hydrogenase (black letter) and formate dehydrogenase (green letter) in cocultures of G. metallireducens-G. sulfurreducens (GS/GM) and G. sulfurreducens/P. carbinolicus (GS/PC). The n-fold change shown at the right represents the gene transcript upregulation or downregulation in G. sulfurreducens in the GS/GM coculture compared to the transcript level in the GS/PC coculture. The n-fold change is presented only for G. sulfurreducens genes with significant expression (log₂ RPKM ≥ 8) in one of the cocultures.

Fig 2

Heat map comparison of expression levels of genes associated with DIET in cocultures. For details, see the legend to Fig. 1.

Fig 3

(a) RT-qPCR assays of selected genes involved in DIET. The y axis shows normalized gene expression values based on the housekeeping gene recA. Standard deviations were calculated from triplicate independent samples. (b) Coomassie-stained 12% SDS-PAGE of equal amounts of protein from cocultures of G. sulfurreducens with G. metallireducens (GS/GM) and P. carbinolicus (GS/PC). (c) Western blot probed for G. sulfurreducens PilA protein.

Fig 4

Heat map comparison of expression levels of genes associated with central metabolism in cocultures. For details, see the legend to Fig. 1.

Fig 5

Central metabolism in G. sulfurreducens showing metabolic pathways when acetate is available. Genes that are upregulated (≥2-fold) in G. metallireducens-G. sulfurreducens cocultures compared to those in P. carbinolicus-G. sulfurreducens are in bold and underlined. Double-headed arrows represent reversible reactions.