Nitrate Reduction Stimulates and Is Stimulated by Phenazine-1-Carboxylic Acid Oxidation by Citrobacter portucalensis MBL.

Derek R Lovley,Lev M Tsypin,Dianne K Newman

doi:10.1128/mbio.02265-21

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 oxidizes the PCA at a rate that is environmentally relevant. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other gammaproteobacteria with various capacities to respire nitrate. We found that PCA oxidation by these strains in a nitrate-dependent manner is decoupled from growth and strain dependent. We infer that bacterial PCA oxidation is widespread and 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.

Highlights

Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation
It was discovered that phenazine reduction can greatly benefit P. aeruginosa by: (i) regulating gene expression in P. aeruginosa during quorum sensing by oxidizing a transcription factor, (ii) acting as alternative terminal electron acceptors to promote anoxic survival, and (iii) facilitating iron acquisition [4,5,6,7,8]
The effect of Phenazine-1-carboxylic acid (PCA) oxidation by C. portucalensis MBL on its rate of nitrate reduction was outsized (Fig. 2A). This is consistent with two explanations. (i) A prior report argues that neutral red oxidation affects electrosynthesis during anaerobic respiration primarily by changing gene regulation via menaquinone reduction [16], and so it is plausible that PCA oxidation may increase transcription of a rate-limiting factor in the electron transport chain to nitrate. (ii) We observed that a PCA redox cycle by C. portucalensis MBL is possible: the cells naturally reduce PCAox, but this is not detectable while nitrate is present, potentially meaning that the PCA is being reoxidized as soon as it is reduced

Summary

Introduction

Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. We enriched for PCA oxidizers from topsoil by incubating them with reduced PCA, acetate (a nonfermentable carbon source), and nitrate as the only terminal electron acceptor, and successfully isolated the PCA-oxidizing Citrobacter portucalensis MBL, which is unable to synthesize its own phenazines [20]. Adding nitrate to the PCAox condition caused reduction to be nondetectable, and the presence of nitrite, which abiotically oxidizes PCAred, caused a slight dampening in the reduction rate by 1.25 6 0.09 mM/h (Fig. S1).

Results

Conclusion