The shewanellae are aquatic microorganisms with worldwide distribution. Their hallmark features include unparalleled respiratory diversity and the capacity to thrive at low temperatures. As a genus the shewanellae are physiologically diverse, and this review provides an overview of the varied roles they serve in the environment and describes what is known about how they might survive in such extreme and harsh environments. In light of their fascinating physiology, these organisms have several biotechnological uses, from bioremediation of chlorinated compounds, radionuclides, and other environmental pollutants to energy-generating biocatalysis. The ecology and biotechnology of these organisms are intertwined, with genomics playing a key role in our understanding of their physiology.

Shewanella oneidensisstrain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling inS. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed withS. oneidensisto identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamedbfe(bacterialflavin adenine dinucleotide [FAD]exporter) based on phenotypic characterization. Deletion ofbferesulted in a severe decrease in extracellular flavins, while overexpression ofbfeincreased the concentration of extracellular flavins. Strains lackingbfehad no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-typeS. oneidensisand the Δbfemutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors,bfemutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates byS. oneidensisand have identified the first FAD transporter in bacteria.IMPORTANCEExtracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacteriumShewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequencedShewanellaspecies that facilitates export of flavin electron shuttles inS. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity inS. oneidensis.

The Mtr respiratory pathway ofShewanella oneidensisstrain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. OtherShewanellaspecies tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH₄⁺) to dinitrogen gas (N₂) using intracellular electron acceptors such as nitrite (NO₂⁻) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH₄⁺ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. ¹⁵N-labeling experiments revealed that NH₄⁺ was oxidized to N₂ via hydroxylamine (NH₂OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH₄⁺ oxidation with electrode as electron acceptor. Complete NH₄⁺ oxidation to N₂ without accumulation of NO₂⁻ and NO₃⁻ was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.

Bioelectrochemical systems rely on microorganisms to link complex oxidation/reduction reactions to electrodes. For example, in Shewanella oneidensis strain MR-1, an electron transfer conduit consisting of cytochromes and structural proteins, known as the Mtr respiratory pathway, catalyzes electron flow from cytoplasmic oxidative reactions to electrodes. Reversing this electron flow to drive microbial reductive metabolism offers a possible route for electrosynthesis of high value fuels and chemicals. We examined electron flow from electrodes into Shewanella to determine the feasibility of this process, the molecular components of reductive electron flow, and what driving forces were required. Addition of fumarate to a film of S. oneidensis adhering to a graphite electrode poised at −0.36 V versus standard hydrogen electrode (SHE) immediately led to electron uptake, while a mutant lacking the periplasmic fumarate reductase FccA was unable to utilize electrodes for fumarate reduction. Deletion of the gene encoding the outer membrane cytochrome-anchoring protein MtrB eliminated 88% of fumarate reduction. A mutant lacking the periplasmic cytochrome MtrA demonstrated more severe defects. Surprisingly, disruption of menC, which prevents menaquinone biosynthesis, eliminated 85% of electron flux. Deletion of the gene encoding the quinone-linked cytochrome CymA had a similar negative effect, which showed that electrons primarily flowed from outer membrane cytochromes into the quinone pool, and back to periplasmic FccA. Soluble redox mediators only partially restored electron transfer in mutants, suggesting that soluble shuttles could not replace periplasmic protein-protein interactions. This work demonstrates that the Mtr pathway can power reductive reactions, shows this conduit is functionally reversible, and provides new evidence for distinct CymA:MtrA and CymA:FccA respiratory units.