Nitrate reduction stimulates and is stimulated by phenazine-1-carboxylic acid oxidation by Citrobacter portucalensis MBL

Abstract

ABSTRACTPhenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate. When C. portucalensis MBL is provided reduced PCA and nitrate, it rapidly oxidizes the PCA. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other γ-proteobacteria with varying capacities to respire nitrate. We found that PCA oxidation by these strains in a nitrate-dependent manner is decoupled from growth and correlated with their possession of the periplasmic nitrate reductase Nap. We infer that bacterial PCA oxidation is widespread and propose that it may be genetically determined. Notably, oxidizing PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric exchange of electrons from PCA to nitrate, which we attribute to C. portucalensis MBL’s ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent.IMPORTANCEPhenazines are increasingly appreciated for their roles in structuring microbial communities. These tricyclic aromatic molecules have been found to regulate gene expression, be toxic, promote antibiotic tolerance, and promote survival under oxygen starvation. In all of these contexts, however, phenazines are studied as electron acceptors. Even if their utility arises primarily from being readily reduced, they need to be oxidized in order to be recycled. While oxygen and ferric iron can oxidize phenazines abiotically, biotic oxidation of phenazines has not been studied previously. We observed bacteria that readily oxidize phenazine-1-carboxylic acid (PCA) in a nitrate-dependent fashion, concomitantly increasing the rate of nitrate reduction to nitrite. Because nitrate is a prevalent terminal electron acceptor in diverse anoxic environments, including soils, and phenazine-producers are widespread, this observation of linked phenazine and nitrogen redox cycling suggests an underappreciated role for redox-active secreted metabolites in the environment.