Divergent Nrf Family Proteins and MtrCAB Homologs Facilitate Extracellular Electron Transfer in Aeromonas hydrophila

Metal-reducing microorganisms are used for electricity production, bioremediation of toxic compounds, wastewater treatment, and production of valuable compounds. Despite numerous microorganisms being reported to reduce metals, the molecular mechanism has primarily been studied in two model systems, Shewanella oneidensis and Geobacter sulfurreducens . We have characterized the mechanism of extracellular electron transfer in Aeromonas hydrophila , which uses the well-studied Shewanella system, MtrCAB, to move electrons across the outer membrane; however, most Aeromonas spp. appear to use a novel mechanism to transfer electrons from the inner membrane through the periplasm and to the outer membrane. The conserved use of MtrCAB in Shewanella spp. and Aeromonas spp. for metal reduction and conserved genomic architecture of metal reduction genes in Aeromonas spp. may serve as genomic markers for identifying metal-reducing microorganisms from genomic or transcriptomic sequencing. Understanding the variety of pathways used to reduce metals can allow for optimization and more efficient design of microorganisms used for practical applications. Extracellular electron transfer (EET) is a strategy for respiration in which electrons generated from metabolism are moved outside the cell to a terminal electron acceptor, such as iron or manganese oxide. EET has primarily been studied in two model systems, Shewanella oneidensis and Geobacter sulfurreducens . Metal reduction has also been reported in numerous microorganisms, including Aeromonas spp., which are ubiquitous Gammaproteobacteria found in aquatic ecosystems, with some species capable of pathogenesis in humans and fish. Genomic comparisons of Aeromonas spp. revealed a potential outer membrane conduit homologous to S. oneidensis MtrCAB. While the ability to respire metals and mineral oxides is not widespread in the genus Aeromonas , 90% of the sequenced Aeromonas hydrophila isolates contain MtrCAB homologs. A. hydrophila ATCC 7966 mutants lacking mtrA are unable to reduce metals. Expression of A. hydrophila mtrCAB in an S. oneidensis mutant lacking homologous components restored metal reduction. Although the outer membrane conduits for metal reduction were similar, homologs of the S. oneidensis inner membrane and periplasmic EET components CymA, FccA, and CctA were not found in A. hydrophila . We characterized a cluster of genes predicted to encode components related to a formate-dependent nitrite reductase (NrfBCD),