Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer

Abstract

Antibiotics are increasingly recognized as having other, important physiological functions for the cells that produce them. An example of this is the effect that phenazines have on signaling and community development for Pseudomonas aeruginosa (L. E. Dietrich, T. K. Teal, A. Price-Whelan, and D. K. Newman, Science 321:1203-1206, 2008). Here we show that phenazine-facilitated electron transfer to poised-potential electrodes promotes anaerobic survival but not growth of Pseudomonas aeruginosa PA14 under conditions of oxidant limitation. Other electron shuttles that are reduced but not made by PA14 do not facilitate survival, suggesting that the survival effect is specific to endogenous phenazines.

FIG. 1.

Representative cyclic voltammetry (CV) of Δphz mutant cultures of P. aeruginosa PA14 incubated anaerobically in 100 ml MOPS medium containing 20 mM glucose, supplemented with 90 μM PYO (dark trace) or no PYO (light trace). PYO is the only electrochemically active component with single anodic (oxidation) and cathodic (reduction) peaks characteristic of itself. CV experiments were performed at 100 mV/s, with electrodes consisting of a stationary gold disk working electrode (BASi), an Ag/AgCl reference electrode, and a Pt counter electrode.

FIG. 2.

PYO (a), PCA (b), and 1-OHPHZ (c) function as electron shuttles (▪) to promote anaerobic survival of the Δphz mutant of P. aeruginosa PA14 when cells are incubated anaerobically in MOPS-buffered medium containing 20 mM d-glucose and ∼90 μM phenazine (PYO, PCA, or 1-OHPHZ) and with the graphite rod working electrode poised at +0.2 V versus that of the NHE. Survival was determined by measuring the number of CFU on LB agar plates. The number of CFU of Δphz anaerobic incubations without phenazine (▿), poised potential (▵), or both (⋄) served as a control. Error bars represent standard deviations from at least triplicate samples in each experimental set. Plots represent results from at least three independent experiments.

FIG. 3.

Anaerobic survival of the Δphz mutant of P. aeruginosa PA14 without d-glucose and in the presence of 20 mM d-glucose for cells incubated in MOPS medium containing ∼90 μM PYO, with the graphite rod working electrode poised at +0.2 V versus that of the NHE. Survival was determined by the number of CFU on LB agar plates. Error bars represent standard deviations from triplicate samples in each experimental set. Plots represent results from two independent experiments.