A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1

Although it has been established that electron mediators substantially promote extracellular electron transfer (EET), electron shuttling pathways are not fully understood. Here, a new electron shuttling pathway was found in the EET process by Shewanella oneidensis MR-1 with resazurin, a lipophilic electron mediator. With resazurin, the genes encoding outer-membrane cytochromes (mtrCBA and omcA) were downregulated. Although cytochrome deletion substantially reduced biocurrent generation to 1-12% of that of wild-type (WT) cells, the presence of resazurin restored biocurrent generation to 168 mu A center dot cm-2 (Delta mtrA/ omcA/mtrC), nearly equivalent to that of WT cells (194 mu A center dot cm-2), indicating that resazurin-mediated electron transfer was not dependent on the Mtr pathway. Biocurrent generation by resazurin was much lower in Delta cymA and Delta mtrA/omcA/mtrC/fccA/cctA mutants (4 and 6 mu A center dot cm-2) than in WT cells, indicating a key role of FccA, CctA, and CymA in this process. The effectiveness of resazurin in EET of Mtr cytochrome mutants is also supported by cyclic voltammetry, resazurin reduction kinetics, and in situ c-type cytochrome spectroscopy results. The findings demonstrated that low molecular weight, lipophilic electron acceptors, such as phenoxazine and phenazine, may facilitate electron transfer directly from periplasmic and inner membrane proteins, thus providing new insight into the roles of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.

Automated summary

A new electron shuttling pathway was discovered in Shewanella oneidensis MR-1 using the lipophilic electron mediator resazurin. This pathway is independent of the Mtr pathway and relies on the involvement of FccA, CctA, and CymA. The findings provide new insights into the role of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.