K-046. Key Components of Central Metabolism Contribute to Pyocyanin Reduction Activity in Pseudomonas aeruginosa

A. Price-Whelan1, J. Huang2, D. K. Newman1;
1Massachusetts Inst. of Technology, Cambridge, MA, 2California Inst. of Technology, Pasadena, CA.

Since the 19th century, physicians have documented the production of blue-pigmented pus and sputum in patients with burn wound and lung infections. The compound responsible for this coloration is pyocyanin, a redox-active phenazine produced by Pseudomonas aeruginosa. The role of pyocyanin in eukaryotic infections has been studied extensively, and specific intracellular reductants and enzymes participate in the redox transformation of pyocyanin in host epithelial cells. P. aeruginosa also catalyzes pyocyanin reduction, however the mechanisms underlying this activity are not well understood. We used the P. aeruginosa PA14 nonredundant library to screen for mutants defective in electron transfer to ferric citrate, a reaction that required pyocyanin as a mediator under our conditions. Out of 305 mutants that were defective in ferric citrate reduction, we chose 109 candidate mutants that contained transposon insertions in genes potentially involved in metabolism or redox homeostasis. These mutants were subjected to a secondary screen for defects in growth or pyocyanin biosynthesis. Mutants with insertions in one of two operons were defective in pyocyanin reduction, with minor defects in growth and no defects in pyocyanin biosynthesis. The two operons we identified were: 1) one composed of the structural genes for the cytochrome bc1 complex of the aerobic respiratory chain, and 2) one encoding the biosynthetic glycerol-3-phosphate dehydrogenase and a putative regulatory protein. These gene products contribute to the establishment of the proton-motive force and maintain the integrity of the membrane, suggesting that pyocyanin respiration and/or active transport contribute to the pyocyanin electron shuttling activity observed in P. aeruginosa cultures.