Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aer uginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2 G2, are homologous to previously studied phenazine biosynthetic operons fro m Pseudomonas fluorescens. and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated th at each of the biosynthetic operons from P. aeruginosa is sufficient for pr oduction of a single compound, phenazine-l-carboxylic acid (PCA). Subsequen t conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes,phzM and phzS, which encode putative phenazine-sp ecific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonp roducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine . P. aeruginosa with insertionally inactivated phzM or phzS developed pyocy anin-deficient phenotypes. A third phenazine-modifying gene, phzH, which ha s a homologue in Pseudomonas chlororaphis, also was identified and was show n to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1 Our results suggest that there is a complex pyocyanin biosynthetic pa thway in P. aeruginosa consisting of two core loci responsible for synthesi s of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carbox amide.