Physiological consequences of long-term PCA oxidation for C. portucalensis MBL.

(A) Diagram of the bioelectrochemical reactor. The C. portucalensis MBL culture was incubated in the main chamber with a working electrode poised to -500 mV that continuously reduced any available PCA. The reference electrode communicated with the potentiostat to retain a constant voltage. Cells oxidized PCA when nitrate was present. In a sidearm, separated by a dense glass frit, the counter-electrode completed the circuit. Reduced PCA is green and oxidized PCA is colorless. (B) Current traces for biological replicates with and without provided PCA and an initial concentration of 10 mM nitrate. Samples were taken from these replicates at the times displayed in (C)-(E), where each data point corresponds to an independent biological replicate. (C) Time point measurements of survival in the culture, as determined by colony forming units (CFUs). The asterisks represent a Bonferroni-corrected statistically significant difference between the two conditions at the final time point (p = 0.0025). (D) The nitrite that was produced (from nitrate reduction) by the cultures over the experiment. At the third and fourth time point there were Bonferroni-corrected statistically significant differences between the two conditions: p = 0.0029 and p = 0.0019, respectively. (E) The normalized ATP content per CFU over the time course. There were no statistically significant differences. The Bonferroni corrected threshold was p < 0.0083 given hypothesis testing across the six time points.</p

Comparison of the roles of the three terminal nitrate reductases in PCA oxidation.

(A-C) Comparison of homologous recombination knockouts versus translational knockouts for each individual terminal nitrate reductase. Each graph shows the oxidation of PCA over time (measured by the decay of PCAred, which is fluorescent) with the abiotic control (nitrate plus reduced PCA in reaction medium without cells) in grey. (A) NarZ comparison: the deletion (Δ) and translational knockout (tlKO) strains all have a PCA oxidation deficit relative to the wildtype (WT, blue). The most severe phenotype is in the ΔnarUZYWV strain (green), and the ΔnarZYWV and narZ-tlKO (yellow and orange, respectively) phenotypes are indistinguishable. (B) NarG comparison: the deletion (green) and translational knockout (orange) strains have the same slight PCA oxidation deficit relative to the wildtype control (WT, blue). (C) NapA comparisons: the translational knockout (orange) has a more severe PCA oxidation deficit that the deletion strain (green), relative to the wildtype control (blue). Each thick line corresponds to the mean of three biological replicates plotted in semitransparent circles. (D-E) Comparisons of the maximum observed PCA oxidation rate for each combinatorial nitrate reductase mutant (and abiotic control). (D) The phenotypes after shaking (oxic) pregrowth. (E) The phenotypes after standing (hypoxic) pregrowth. (F-G) Comparisons of the time until half of the PCA was oxidized for each combinatorial nitrate reductase mutant (and abiotic control). (F) The phenotypes after oxic pregrowth. (G) The phenotypes after hypoxic pregrowth. N.D. stands for “not detected,” corresponding to strains that did not reach the 50% PCA oxidized threshold over the 48-hour assay. Squares represent the means of technical triplicates and circles are independent biological replicates with no technical replicates. Double asterisks represent statistical significance after Bonferroni correction, and single asterisks represent p S1 Text), and the complete matrices of pairwise comparisons are presented in S2 Fig. The conversion of PCA oxidation curves to rate and time metrics is described in S3 Fig.</p

Nitrate-driven PCA oxidation rates in quinone knockout backgrounds, including single nitrate reductase strains.

(A) Maximum PCA oxidation rates. Squares represent the means of technical triplicates and circles represent independent biological replicates. (B) Pairwise comparisons between differences of mean maximum PCA oxidation rates for the strains in (A). Given 28 comparisons, the Bonferroni-corrected threshold for significance is p < 0.00179.</p

Oligos for building complementation vectors.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the 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.</div

Oligos for Datsenko-Wanner deletions.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the 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.</div

Model for how phenazine redox cycling may be mutualistic or competitive in microbial communities across redox gradients.

(A) For a bacterium that finds itself starved for its preferred terminal electron acceptor (yellow cell under reducing stress), oxidized PCA can serve as an alternative. For a bacterium that finds itself starved for its preferred electron donor (cyan cell under oxidative stress), reduced PCA can serve as an alternative. Together, the two cells can more efficiently use bioavailable nutrients with the PCA redox cycle as a bridge. This scheme would work best if the phenazine oxidizer has exclusive access to the terminal electron acceptor and that the phenazine reducer has exclusive access to the terminal electron donor. (B-C) For a bacterium that relies on the abiotic oxidation of PCA (yellow cell, B) to release insoluble nutrients from iron minerals such as ferric oxyhydroxides to which phosphate is bound, the presence of a PCA oxidizing species would starve it (cyan cell, C). This situation could occur if the environment contains a TEA that the blue cell can respire, but the yellow cell cannot, and the rate of PCA oxidation by the blue cell exceeds its abiotic oxidation rate by iron minerals. A specific example could pair a P. aeruginosa (as the yellow cell) and C. portucalensis MBL (as the cyan cell) in the presence of fumarate [23].</p

Models and thermodynamics of PCA oxidation.

(A) Potential models of PCA oxidation in Gram-negative bacteria capable of respiration. In aqueous environments, PCA redox reactions are two-electron, two-proton processes. Reduced PCA is shown in green, reflecting its true color. PCA oxidation can theoretically be coupled to the respiratory electron transport chain in a couple ways: (Category 1, electron transfer to terminal reductase) PCA may donate electrons to the terminal reductase (grey shape) for a respirable terminal electron acceptor (T.E.A.), thus contributing two protons to the periplasm; (Category 2, electron transfer to quinol pool) PCA may donate two electrons and two protons to quinones, thus regenerating the quinol pool. (Category 3, electron transfer to terminal electron acceptor) Alternatively, PCA directly reduces the terminal electron acceptor. This may happen externally to the cell, or PCA may enter the cytoplasm and react with the terminal electron acceptor independently of the electron transport chain, as depicted. In this illustration, the arrows may represent direct reactions or ones mediated by enzymes or other factors. Transferring electrons to the terminal reductase or quinol pool (Categories 1 and 2) represents scenarios that may be energetically beneficial for a respiring bacterial cell, whereas transferring electrons to the terminal electron acceptor (Category 3) may be detrimental. This would require the transport of PCA across the inner membrane because its carboxylic acid moiety is negatively charged at circumneutral pH, and it cannot passively cross the membrane. For simplicity, this illustration does not show potential reactions with a periplasmic reductase, but the logic would remain the same, only with no involvement of the cytoplasmic space. (B) Electron tower of relevant half-reactions. Reactions are ordered by their relative standard midpoint potentials with more negative values on top and more positive ones on the bottom (not to scale). Thermodynamically favorable pairings comprise more positive half-reactions with more negative ones in reverse. The theoretical limit for energy that can be conserved from a pairing correlates with the magnitude of the difference in half-reaction potentials. PCA: phenazine-1-carboxylic acid. MQ: menaquinone. DMQ: demethylmenaquinone. UQ: ubiquinone. Fum2-: fumarate. Succ2-: succinate. TMAO: trimethylamine-N-oxide. TMA: trimethylamine. DMSO: dimethyl sulfoxide. DMS: dimethyl sulfide. NO2-: nitrite. NO: nitric oxide. NO3-: nitrate.</p

All pairwise comparisons for the strains in Fig 6.

(A) Statistical significance matrix for the maximum PCA oxidation rate in the presence of fumarate. (B) Statistical significance matrix for the maximum PCA oxidation rate in the presence of DMSO. (C) Statistical significance matrix for the maximum PCA oxidation rate in the presence of TMAO. (TIF)</p

Strains used in this study.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the 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.</div

Explicit conceptual model of PCA oxidation during anaerobic nitrate respiration.

To interpret PCA oxidation phenotypes during anaerobic nitrate respiration, it is necessary to keep in mind the distinct electron pathways in the respiratory (A) and periplasmic (B) nitrate reductase complexes. These models are made assuming a complete analogy to the arrangement of these proteins in E. coli [25, 29, 30]. The paths of the electrons along the reductase complexes are not shown for simplicity, but they flow from the quinols to the nitrate in each case. The categories of PCA interactions are numbered according to the scheme in Fig 1A and Table 1. In the case of the respiratory nitrate reductase complexes, there are two redundant homologs in C. portucalensis: NarGHI and NarZYV [42]. NarI/V can accept electrons from all three types of quinones [25]. For the periplasmic nitrate reductase, there are two distinct quinone interaction sites (NapH for ubiquinone and NapC for menaquinone); notably, demethylmenaquinone does not appear to play a role in periplasmic nitrate reductase activity [30]. The NapAB complex is soluble in the periplasmic space, and the electrons from the NapHGC complex are ferried to NapA by NapB, which is a cytochrome c-type protein [29]. Category 1 (electron transfer to terminal reductase) PCA interactions are depicted as occurring at quinol-oxidizing subunits of the protein complexes (NarI, NarV, NapH, and NapC) to illustrate the hypothesis that a reduced PCA molecule may replace a quinol. Auxiliary and chaperone proteins that are members of the nitrate reductase operons and are involved in complex formation but not activity (NarJ, NarW, NapF, and NapD) are not illustrated [29]. Note: P. aeruginosa possesses only one set of homologs for the respiratory nitrate reductase (NarGHI) and the periplasmic nitrate reductase.</p