A seven-gene locus for synthesis of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2-79

Abstract

Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kb BglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all phz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the phzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5'-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the phzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes, phzX and phzY, that are homologous to phzA and phzB, respectively, were cloned and sequenced from P. aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.

FIG. 1

Physical maps of cosmid clone pPHZ108 (A), the DNA region from P. fluorescens 2-79 encoding genes involved in the production of PCA (B), and a portion of the homologous genetic locus from P. aureofaciens 30-84 (C). Open boxes indicate genes encoding phenazine biosynthesis enzymes. The direction of the gene transcription is shown by an arrow. The symbols P_A and P_X represent the position and orientation of corresponding promoters. Insertions of Tn3-lacZ that interfered with phenazine production are marked on the map of pPHZ108A as •| (Lac⁺ phenotype) and ○| (Lac⁻ phenotype).

FIG. 2

Alignment of the deduced amino acid sequences of the PhzA (P. fluorescens 2-79), PhzX (P. aureofaciens 30-84), PhzB (P. fluorescens 2-79), and PhzY (P. aureofaciens 30-84) proteins. Identical (∗) and similar (·) amino acid sequences are indicated. Dashes represent gaps inserted in the sequences to improve the alignment.

FIG. 3

Autoradiograph of polypeptides labeled with [³⁵S]methionine and resolved by electrophoresis on an SDS–12% polyacrylamide gel. Samples were prepared from E. coli BL21 containing pGP1-2 plus the indicated plasmids. The location of DNA fragments inserted in pT7-5 and pT7-6 vectors for gene expression is shown in Fig. 4. Lanes: 1, pT7-6; 2, pT7-6ABCD; 3, pT7-6AB; 4, pT7-5CDE; 5, pT7-6CDEFG; 6, pT7-5CD; 7, pT7-5G. An intense 16-kDa band in lane 4 is a product of a truncated phzF gene, and an intense protein band almost overlapping with PhzC in lane 2 is a product of a truncated phzE gene. Positions of molecular size markers are given on the left in kilodaltons.

FIG. 4

Analysis of phenazine biosynthesis in P. fluorescens 2-79 gene replacement mutants (A) and by E. coli BL21(DE3) clones harboring plasmids with different sets of phz genes from P. fluorescens 2-79 (B) or P. aureofaciens 30-84 (C) cloned under the control of a T7 promoter. Restriction maps, locations of individual phz genes, and DNA fragments contained within plasmids used in the study are shown. Arrows indicate the position and orientation of the T7 promoter. Solid triangles denote locations of the Kan^r cassette insertions in the chromosome of P. fluorescens 2-79 mutants. N.D., not detected. OH-PCA, 2-hydroxyphenazine-1-carboxylic acid; OH-PHZ, 2-hydroxyphenazine.

FIG. 5

Proposed action of PhzC, PhzD, PhzE, PhzF, and PhzG in the biosynthesis of PCA.