Flavin electron shuttles dominate extracellular electron transfer by Shewanella oneidensis

Abstract

Shewanella oneidensis strain 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 in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had 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-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant 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 by S. oneidensis and have identified the first FAD transporter in bacteria.

Importance: Extracellular 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 bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. 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 in S. oneidensis.

FIG 1

Flavin profile of S. oneidensis (SO) or E. coli (EC) strains quantified by HPLC. S. oneidensis cultures were anaerobically grown in SBM with 20 mM lactate and 40 mM fumarate at 30°C. Balch tubes were made anaerobic by flushing nitrogen gas through butyl rubber stoppers for 15 min. After 15 h of incubation, a sample was taken and cells were removed by centrifugation. HPLC was performed as previously described (19). The ΔushA E. coli strain was grown in SBM with 20 mM lactate overnight at 37°C. Error bars indicate SEM (n = 3).

FIG 2

Electron shuttles accelerate reduction of insoluble extracellular electron acceptors. (A) Fe(III) oxide (ferrihydrite) reduction was quantified as previously described (13) for the following strains: MR-1 + vector (●), MR-1 + bfe (○), Δbfe strain + vector (▾), and Δbfe strain + bfe (▽). Error bars indicate SEM (n = 3). (B) Bioreactors were assembled as previously described (9). One milliliter from an aerobic SBM culture with 20 mM lactate was added to 9 ml of an anaerobic SBM culture with 50 mM lactate and 40 mM fumarate. Cultures were grown at 30°C with shaking until an optical density at 600 nm of 0.4 was reached. The entire culture was added to the bioreactor. Bioreactors were continuously flushed with nitrogen gas, and electrodes were poised at a potential of +0.242 V versus a standard hydrogen electrode using a 16-channel VMP potentiostat (Bio-Logic SA). Current measurement of MR-1 (black), MR-1 + 10 µM FMN (flavin mononucleotide) (gray), the Δbfe mutant (blue), and the Δbfe mutant + 10 µM FMN (red) in bioreactors is shown. Data are representative of three replicates.