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Title: FUNCTIONAL ANALYSIS OF GENES FOR BIOSYNTHESIS OF PYOCYANIN AND PHENAZINE-1-CARBOXAMIDE FROM PSEUDOMONAS AERUGINOSA PAO1

Author
item MAVRODI, DMITRI - WASHINGTON STATE UNIV.
item BONSALL, ROBERT - WASHINGTON STATE UNIV.
item DELANEY, SHANNON - WASHINGTON STATE UNIV.
item SOULE, MARILYN - WASHINGTON STATE UNIV.
item Phillips, Greg
item Thomashow, Linda

Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2001
Publication Date: 11/1/2001
Citation: Mavrodi, D.V., Bonsall, R.F., Delaney, S.M., Soule, M.J., Phillips, G., Thomashow, L.S. 2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from pseudomonas aeruginosa pao1. Journal of Bacteriology. 183.21.6454-6465.2001

Interpretive Summary: Certain strains of root-colonizing fluorescent Pseudomonas bacteria have gained attention in recent years because they produce broad-spectrum metabolites active against fungal pathogens that cause soilborne root diseases. One such class of compounds, the phenazines, includes over 50 members with the same core structure but with different chemical modifications that largely determine their physical properties and biological activity. In P. aeruginosa, the most widely-studied phenazine-producing pseudomonad, phenazines contribute not only to biological control activity but also to the activity of the bacteria as opportunistic pathogens of animals, insects, nematodes, and plants. Although the physiological effects of phenazines produced by P. aeruginosa on host organisms are well known, the biochemistry and genetics of phenazine synthesis in P. aeruginosa have remained unclear. Here we show that P. aeruginosa PAO1 contains a complex phenazine biosynthetic pathway consisting of two conserved sets of "core" biosynthetic genes responsible for the synthesis of phenazine-1-carboxylic acid (PCA) and three additional genes that encode unique enzymes involved in the conversion of PCA to other phenazines, pyocyanin and phenazine-1-carboxamide. A common core biosynthetic pathway was shown to be conserved among phenazine-producing nonpathogenic fluorescent Pseudomonas spp. but was not detected in other phenazine-nonproducing bacterial genera. Knowledge of the genes responsible for phenazine product specificity could reveal ways to manipulate the bacteria to produce multiple phenazines or hybrid compounds not previously described. Such compounds may have improved activity against soilborne plant pathogens or lead to the development of novel pharmaceutical products.

Technical Abstract: Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from P. fluorescens and P. aureofaciens. Functional studies in phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, that encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase enzymes, respectively. Expression of phzS alone in E. coli or in pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS genes developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in P. chlororaphis, also was identified and shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest the existence of a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA, and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine and phenazine-1-carboxamide.