Independently evolved extracellular electron transfer pathways in ecologically diverse Desulfobacterota

Extracellular electron transfer plays a role in the biogeochemical cycling of carbon, metals, sulfur, and nitrogen, and has wide-ranging biotechnological applications. The metal-reducing (Mtr), outer-membrane cytochrome (Omc), and porin-cytochrome (Pcc) pathways facilitate electron transfer to insoluble electron acceptors via trans-outer membrane cytochrome complexes. Although these pathways perform a similar function, they are phylogenetically unrelated, indicating independent evolutionary origins. Here, we report an extracellular electron transfer mechanism in which the high-current producing bacterium Desulfuromonas acetexigens differentially co-expresses, at transcript and protein levels, the porin-cytochrome, outer-membrane cytochrome, and metal-reducing pathways, along with high-molecular-weight cytochromes containing a large number of hemes (up to 86 heme-binding motifs), under extracellular electron transfer growth conditions (i.e. electrode under set potential or naturally occurring iron oxide minerals as the electron acceptor). Additionally, we identified over 40 Desulfobacterota species from diverse ecological environments that encode the outer-membrane cytochrome and metal-reducing pathways, with the majority also expressing the porin-cytochrome pathway. The newly identified metal-reducing proteins in Desulfobacterota form a major lineage, greatly expanding the known diversity of these proteins. To our knowledge, mtrCAB genes have not been reported in the Desulfobacterota phylum (formerly classified as Deltaproteobacteria), nor has any electroactive organism been shown to express these phylogenetically distant pathways simultaneously. These findings have ecological implications, challenging the belief that certain extracellular electron transfer pathways are exclusive to specific taxa, and suggesting that these pathways are more widespread than previously thought. Additionally, this reveals a previously unrecognized versatility in microbial electron transfer mechanisms that can be exploited in biotechnological applications.