Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force

Abstract

While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton-motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox-active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation.

Figure 1

Metabolic pathways in P. aeruginosa. Multiple arrowheads indicate that several reaction steps have been condensed for clarity. Boxes indicate the enzyme that catalyzes each reaction. (A) Glucose oxidation via the Entner-Doudoroff pathway. For clarity, the pathway is abbreviated to the key intermediates of glycolysis. (B) Pyruvate fermentation (Eschbach et al., 2004). LdhA, lactate dehydrogenase; Pdh, pyruvate dehydrogenase; Pta, phosphate acetyltransferase; AckA, acetate kinase. (C) The arginine deiminase pathway (Vander Wauven et al., 1984). ArcA, arginine deiminase; ArcB, ornithine carbamoyltransferase; ArcC, carbamate kinase.

Figure 2

Phenazine redox cycling in P. aeruginosa PA14 ∆phz1/2. Cells were grown aerobically in LB to an OD₅₀₀ of 2.8 and then pelleted, washed, and resuspended in anoxic MOPS-buffered minimal medium with 20 mM glucose. Where noted in the ‘potential’ column of the legend, cultures also included 75 µM PCA and an electrode poised at a potential to oxidize any reduced PCA. Results are typical of at least three independent experiments. (A) Survival of strains with deletions of pyruvate metabolism genes. 100% survival represents approximately 7 × 10⁸ CFU/mL. (B) HPLC traces from culture supernatants after three days of phenazine cycling as in panel A. Peaks were identified by comparing retention times to known standards. The dashed gray arrow indicates the retention time of lactate, which was not observed in this experiment. Question marks (?) indicate unidentified products. Asterisks (*) indicate trace contaminants that were present from the start of the experiment.

Figure 3

Pyruvate fermentation in the PA14 ∆phz1/2 ∆ldhA strain with 40 mM pyruvate. Cultures were incubated without phenazines or an oxidizing potential (open squares) or with 75 µM PCA and an oxidizing potential (closed squares). Error bars indicate the standard error of three independent experiments. (A) Survival of the ∆phz1/2 ∆ldhA strain during pyruvate fermentation. 100% survival represents approximately 1.4 × 10⁹ CFU/mL. (B) Acetate produced during pyruvate fermentation in the ∆phz1/2 ∆ldhA strain. The amount of acetate produced in each experiment was normalized to the CFU measurement at day 0. Error bars are present but smaller than the data points. (C) ATP concentrations during pyruvate fermentation in the ∆phz1/2 ∆ldhA strain.

Figure 4

Survival with pyruvate fermentation in P. aeruginosa. Where shown, error bars indicate the standard error of three independent experiments. (A) Anaerobic survival for wild type PA14 (WT), PA14 ΔldhA (ΔldhA), PA14 ΔackA (ΔackA), and PA14 ΔackA-pta (ΔackA-pta) in the presence of 40 mM pyruvate (+pyruvate, closed points). WT PA14 without pyruvate is shown for comparison (−pyruvate, open points). 100% survival represents approximately 4 × 10⁸ CFU/mL. Results are typical of least three independent experiments. (B) The [NADH]/[NAD⁺] ratio after 24 hours of pyruvate fermentation in PA14 WT, ∆ldhA, and ∆ackA. (C) ATP concentrations from the same experiments shown in panel B.

Figure 5

Survival with the arginine deiminase pathway in P. aeruginosa. Where shown, error bars indicate the standard error of three independent experiments (A) Anaerobic survival for wild type PA14 (WT) and PA14 arcC::MAR2×T7 (arc::) in the presence of 40 mM arginine. WT PA14 without arginine is shown for comparison (−arginine, open points). 100% survival represents approximately 4 × 10⁸ CFU/mL. Results are typical of at least three independent experiments. (B) The [NADH]/[NAD⁺] ratio after 24 hours of anaerobic incubation with arginine in PA14 WT and arcC::MAR2×T7. (C) ATP concentrations from the same experiments shown in panel B.

Figure 6

Response of P. aeruginosa PA14 to drugs that disrupt the proton-motive force. Error bars represent the standard error of at least three independent experiments. 100% survival represents approximately 4 × 10⁸ CFU/mL. (A) Anaerobic survival of P. aeruginosa PA14 after two days of anaerobic incubation with 40 mM pyruvate. At the start of anaerobiosis, potassium nitrate (50 mM), DCCD (2 mM), and/or CCCP (100 µM) were added as indicated. DCCD and CCCP were dissolved in ethanol, and so as a control ethanol was added to a final concentration of 0.2% where indicated. A culture incubated without pyruvate is shown for comparison (−pyruvate). (B) Anaerobic survival after two days of anaerobic incubation with 40 mM arginine. At the start of anaerobiosis, ethanol (0.2%), DCCD (2 mM), or CCCP (100 µM) were added as indicated. (C) Effect of DCCD on membrane polarization. After 24 hours of anaerobiosis, 2 mM DCCD was added to cultures surviving anaerobically on 40 mM pyruvate (+DCCD, open circles). Since DCCD was dissolved in ethanol, an equivalent concentration of ethanol (0.2%) was added to control cultures (+ethanol, closed circles). Membrane polarization was monitored over time as described in Methods (for an example analysis, see Supplemental Figure 5). Time 0 indicates the addition of DCCD and ethanol to the cultures.

Figure 7

A metabolic model of survival in P. aeruginosa. The ATP synthesized from glycolysis (1), pyruvate fermentation (2), and the arginine deiminase pathway (3) is used for essential processes including transcription, translation, and proteolysis (4). The F₁F_O-ATPase complex hydrolyses ATP to translocate protons across the inner membrane (5), thereby generating a proton-motive force. Excess reducing equivalents from pyruvate oxidation can be dispensed by converting pyruvate to lactate (6). Phenazines can also regenerate oxidants for metabolism (7); although NAD(P)H is a possible electron donor for phenazines in vivo (Price-Whelan et al., 2007), the dashed lines indicate that the nature of this interaction has not been elucidated. Phenazines are then oxidized extracellularly (8) and can be reused. Nitrate reductase can reduce nitrate to nitrite using electrons from the quinone pool (9), permitting oxidation of NADH via NADH dehydrogenase (10), which can result in proton translocation depending on which dehydrogenase complex is used.