Lack of specificity in Geobacter periplasmic electron transfer

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized for menaquinone oxidation depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, separate periplasmic carriers could be needed for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, the role of ppcA and its four paralogs was examined. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower levels than PpcA in the periplasm. Mutants containing only PpcC and PpcE showed defects, but were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c -type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple Δ ppcABCDE Δ pgcA mutants still grew near wild type rates and displayed normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter , and suggests an as-yet undiscovered periplasmic mechanism supports electron transfer to electrodes. Importance Many inner and outer membrane redox proteins used by Geobacter for electron transfer to extracellular acceptors are known to have specific functions. However, how these are connected by periplasmic redox carriers remains poorly understood. Since Geobacter sulfurreducens contains multiple paralogous triheme periplasmic cytochromes, each with their own unique biochemical properties and expression profiles, it has been hypothesized that each cytochrome is involved in different respiratory pathways depending on redox potential or energy conservation needs. Here we show that instead of being specific for single conditions, the many periplasmic cytochromes of Geobacter show evidence of being highly promiscuous. Surprisingly, while any one of 6 triheme cytochromes could support similar growth with soluble or insoluble metals, none of these were required when cells utilized electrodes. These findings could simplify construction of synthetic electron transfer pathways.