ABSTRACTAt least five gene clusters in theGeobacter sulfurreducensgenome encode putative “electron conduits” implicated in electron transfer across the outer membrane, each containing a periplasmic multihemec-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single-gene-cluster deletions and all possible multiple-deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III) and Mn(IV) oxides, and graphite electrodes poised at +0.24 V and −0.1 V versus the standard hydrogen electrode (SHE). Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously describedomcBCcluster caused defects, but deletion of additional components in an ΔomcBCbackground, such asextEFG, were needed to produce defects greater than 50% compared to findings with the wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCDmutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III) oxide, Mn(IV) oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than the wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth conditions showed all of theseextclusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing onlyextABCDdetected no significant changes in expression of genes encoding known redox proteins or pilus components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth ofG. sulfurreducens, depending on the available extracellular electron acceptor.IMPORTANCEGram-negative metal-reducing bacteria utilize electron conduits, chains of redox proteins spanning the outer membrane, to transfer electrons to the extracellular surface. Only one pathway for electron transfer across the outer membrane ofGeobacter sulfurreducenshas been linked to Fe(III) reduction. However,G. sulfurreducensis able to respire a wide array of extracellular substrates. Here we present the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors. Multiple conduit gene clusters appear to have overlapping roles, including two that have never been linked to metal reduction. Another recently described cluster (ExtABCD) was the only electron conduit essential during electrode reduction, a substrate of special importance to biotechnological applications of this organism.
The Vps/VacJ ABC Transporter Is Required for Intercellular Spread of Shigella flexneri