Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation

Abstract

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa. Author summaryMany bacteria have extremely flexible metabolisms, and we are only beginning to understand how they manifest in the environment. Our study focuses on the role of phenazine-1-carboxylic acid (PCA), a molecule that some bacteria synthesize and secrete into their surroundings. PCA is an "extracellular electron shuttle," a molecule that readily transfers electrons between cells and oxidizing/reducing compounds or other cells. Until our investigation, the role of PCA electron-shuttling had only been studied in one direction: how it takes electrons away from cells, and the effect this has on their viability. Here we present a detailed account of the opposite process and its mechanism: what happens when PCA delivers electrons to cells? Our findings indicate that this previously underappreciated process is generalizable to any anaerobically respiring bacterium. Consequently, we expect that electron donation by PCA is widespread in environments where PCA is plentiful and oxygen is sparse, such as in some agricultural soils. The universality of the extracellular electron shuttle oxidation mechanism we describe for PCA suggests that it should also occur with similar small molecules, of which there are thousands, deepening the implication that this is a significant process in the environment and motivating further research into its consequences.