Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

Abstract

Phenazines have long been recognized for their redox properties. While most attention concerning their redox activity has focused on their role in generating reactive oxygen species in the context of infection (13,14,19,22), as early as 1931, Friedheim hypothesized that phenazine reduction might benefit producer cells as an alternative respiratory pigment (12). Several years ago, our group suggested that the context in which this might be most important would be in biofilms, where cell densities are high and access to oxidants is limited (16,31); consistent with this, we recently showed that mutants unable to produce phenazines are defective in community development (8). While this phenotype is likely due to many factors, including a signaling function for phenazines in later stages of growth (9), given the 1931 hypothesis by Friedheim (12) and our related recent work demonstrating that phenazines control redox homeostasis in Pseudomonas aeruginosa (30), we reasoned that phenazines might contribute to the survival of cells experiencing oxidant limitation.As a first step toward testing this, we investigated the effect of redox-active small molecules on anaerobic survival of P. aeruginosa PA14 in stationary-phase planktonic cultures. We justified beginning with planktonic cultures rather than biofilms based on previous studies which have suggested that cells in stationary-phase planktonic culture physiologically resemble cells in established biofilms (15,35,38). Moreover, by working with cells in planktonic cultures, we could build on voltammetric methods that had previously been used to determine how metabolism changes in Escherichia coli in the presence of the synthetic redox-shuttle ferricyanide (36). Similar voltammetric approaches have also been used to study how phenazines (32, 33) and structurally related flavins (23) mediate power generation by microbial fuel cells.We assembled bulk electrolysis-based glass bioreactors housed within an O 2 -and H 2 -free glove box (MBraun) and controlled by a multichannel potentiostat (series G 300; Gamry) outside the glove box. Each bioreactor held a graphite rod working electrode (Alfa Aesar) with an operating surface area of 6 cm 2 , a Ag/AgCl reference electrode (RE-5B; BASi) with a constant potential of ϩ207 mV versus that of the normal hydrogen electrode (NHE), and about 100 ml MOPS (morpholinepropanesulfonic acid) culture medium (100 mM MOPS at pH 7.2, 93 mM NH 4 Cl, 43 mM NaCl, 2.2 mM KH 2 PO 4 , 1 mM MgSO 4 , 5.0 M FeCl 3 ) (modified from reference 29). The bioreactor was joined by a fritted glass junction to a small side chamber, in which a Pt counter electrode made from Pt mesh (Alfa Aesar) soldered to a copper wire completed the circuit. In order to selectively examine different redox-active small molecules, we used the phenazine-null mutant of PA14 (⌬phz) which is deleted in its two phenazine biosynthetic operons (9).We began by focusing on three endogenous phenazinespyocyanin (PYO), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine (1-OHPHZ)-that are ...

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

“…Surprisingly, the ΔphzS colony biofilm, which produced 5-Me-PCA in addition to PCA and PCN, showed a wild-type morphology. This result was unexpected because the product of PhzS, pyocyanin, is the best studied P. aeruginosa phenazine and supports survival in liquid-culture models under oxygenlimited conditions considered relevant for biofilms (17,46). The ΔphzS colony morphology phenotype observed here reveals the importance of 5-Me-PCA production in biofilm morphogenesis.…”

“…1D) (19,37). As phenazines have been shown to support intracellular redox balancing and survival in electron acceptorlimited PA14 cultures (17,46), we interpret the Δphz phenotype as a response to oxidant limitation that increases the colony surface-to-volume ratio and access to oxygen for resident cells. Phenazines in wild-type biofilms that accept electrons from cells in anoxic biofilm subzones could (i) diffuse into oxic zones to react with oxygen and/or (ii) transfer electrons to other phenazines repeatedly until they are ultimately transferred to oxygen in the oxic zone.…”

“…The opportunistic pathogen P. aeruginosa produces phenazines, antibiotics that contribute to virulence not only by directly damaging host tissues but also by enhancing pseudomonad fitness during host colonization (18,21,22,46,55). In particular, P. aeruginosa is well known for production of the bright blue phenazine pyocyanin, which contributes to the coloration of sputum and pus associated with infections.…”

Morphological optimization for access to dual oxidants in biofilms

“…For example, phenazines are a community resource produced at a cost. Once excreted, these compounds may be taken up and participate in repeated redox cycling by both phenazineproducing and nonproducing cells (32). In the biofilm context, strategies may be used to avoid the likely benefit experienced by cheaters that catalyze redox cycling without contributing to the phenazine pool.…”

“…We have proposed that phenazines attenuate this mechanism because they act as electron acceptors for cells in anoxic regions of the biofilm and shuttle electrons to the well-aerated regions, allowing cells to balance their internal redox state (28,32,33). We proposed that a reduced cellular redox status triggers a morphological change at the population level.…”