ABSTRACTExtracellular 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 oneidensisandGeobacter sulfurreducens. Metal reduction has also been reported in numerous microorganisms, includingAeromonasspp., which are ubiquitousGammaproteobacteriafound in aquatic ecosystems, with some species capable of pathogenesis in humans and fish. Genomic comparisons ofAeromonasspp. revealed a potential outer membrane conduit homologous toS. oneidensisMtrCAB. While the ability to respire metals and mineral oxides is not widespread in the genusAeromonas, 90% of the sequencedAeromonas hydrophilaisolates contain MtrCAB homologs.A. hydrophilaATCC 7966 mutants lackingmtrAare unable to reduce metals. Expression ofA. hydrophila mtrCABin anS. oneidensismutant lacking homologous components restored metal reduction. Although the outer membrane conduits for metal reduction were similar, homologs of theS. oneidensisinner membrane and periplasmic EET components CymA, FccA, and CctA were not found inA. hydrophila. We characterized a cluster of genes predicted to encode components related to a formate-dependent nitrite reductase (NrfBCD), here named NetBCD (forNrf-likeelectrontransfer), and a predicted diheme periplasmic cytochrome, PdsA (periplasmicdihemeshuttle). We present genetic evidence that proteins encoded by this cluster facilitate electron transfer from the cytoplasmic membrane across the periplasm to the MtrCAB conduit and function independently from an authentic NrfABCD system.A. hydrophilamutants lackingpdsAandnetBCDwere unable to reduce metals, while heterologous expression of these genes could restore metal reduction in anS. oneidensismutant background. EET may therefore allowA. hydrophilaand other species ofAeromonasto persist and thrive in iron- or manganese-rich oxygen-limited environments.IMPORTANCEMetal-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 oneidensisandGeobacter sulfurreducens. We have characterized the mechanism of extracellular electron transfer inAeromonas hydrophila, which uses the well-studiedShewanellasystem, MtrCAB, to move electrons across the outer membrane; however, mostAeromonasspp. 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 inShewanellaspp. andAeromonasspp. for metal reduction and conserved genomic architecture of metal reduction genes inAeromonasspp. 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.