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-kbBglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to allphz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of thephzC, 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 thephzA 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, phzXand phzY, that are homologous to phzA andphzB, 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.

Roles of the Gac‐Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30‐84

“…The P. chlororaphis subspecies aureofaciens 30-84 genome sequence was recently deposited in GenBank (GenBank Accession #: PRJNA67533). The P. chlororaphis 30-84 phenazine biosynthetic operon contains seven conserved biosynthetic genes phzXYFABCD (Pierson et al 1995;Mavrodi et al 1998), which correspond to phzABCDEFG (according to the P. fluorescens nomenclature, Mavrodi et al 1998). Here, we use the P. chlororaphis nomenclature to conform to the original literature.…”

Phenazines and their role in biocontrol by Pseudomonas bacteria

“…The phenazine biosynthetic operons of P. fluorescens 2-79 and P. chlororaphis PCL1391 contain the phzABCDEFG genes. The nucleotide sequences of P. fluorescens 2-79 (Mavrodi et al ., 1998), P. aureofaciens 30 -84 (Pierson III et al ., 1995), P. chlororaphis PCL1391, and P. aeruginosa PAO1 (Stover et al ., 2000) are very homologous (70 -95% identity). In P. aeruginosa PAO1, two complete sets of phzABCDEFG genes that are 99% homologous have been identified (Stover et al ., 2000).…”

Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of rhizoctonia root rot and in situ antibiotic production

“…Standard methods were used for DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation [27]. The phenazine biosynthetic operon phzABCDEFG from P. fluorescens 2-79, cloned previously as pT7-6A-G [16], was excised as a 6.7-kb BglII-XbaI DNA fragment and introduced into pALTER-Ex1 (Promega, Madison, WI) distal to the tac Tn5-based delivery plasmid with Kan r ; bla oriR6K mobRP4 tnp* gene of Tn5-IS50 R [30] pT7-6A-G pT7-6 containing 6.9 kb BglII-XbaI fragment from pPHZ108A with phzABCDEFG genes from strain 2-79 [16] pUTKm-phz pUTKm containing phzABCDEFG genes under control of the tac promoter This study a bla, b-lactamase; Kan r , kanamycin resistance; Tet r , tetracycline resistance; Rif r , rifampin resistance; Nal r , nalidixic acid resistance; Phz þ , production of phenazine-1-carboxylic acid; Phl þ , production of 2,4-diacetylphloroglucinol.…”

“…Cultures were grown with constant shaking at 27°C in modified KMB broth. Antibiotics were extracted and viable counts were determined at8,16,24,32, 40, and 48 h as described in Section 2. The strains are Q8r1-96 ðjÞ; Z30-97 ðMÞ; Z32-97 (X); Z33-97 ðÃÞ; Z34-97 ðsÞ; and 2-79 ð}Þ.…”