Introduction of the phzH Gene of Pseudomonas chlororaphis PCL1391 Extends the Range of Biocontrol Ability of Phenazine-1-Carboxylic Acid-Producing Pseudomonas spp. Strains

Abstract

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens

“…2A). Each domain also holds a short ␤-strand (␤N and ␤C) that is not part of the ␤-barrel and a short 3 10 -helix ( 1 and 2). The order and orientation of the core secondary structural elements are the same in both domains if the second ␤-strand of the N-terminal domain (␤2) is superimposed on the first ␤-strand of the C-terminal domain (␤8).…”

“…(7)(8)(9)(10). PhzC, PhzD, and PhzE are similar to enzymes of the shikimic acid pathway and, together with PhzF, are absolutely required for phenazine synthesis.…”

Activation of thephzOperon ofPseudomonas fluorescens2-79 Requires the LuxR Homolog PhzR,N-(3-OH-Hexanoyl)-l-Homoserine Lactone Produced by the LuxI Homolog PhzI, and acis-ActingphzBox

“…These secondary metabolites exhibit antimicrobial activity against a wide range of prokaryotic and eukaryotic microbes, and the synthesis and secretion of these compounds is thought to confer an advantage on the producer bacteria when in competition with other microflora in the soil habitat (29). Production of substituted phenazines is also required by these bacteria to fully protect plants against diseases caused by fungal and bacterial pathogens that are susceptible to the antibiotics (2,7,9,33,49). Such biocontrol properties are of considerable interest, since their application in the field could reduce reliance on the use of environmentally toxic antimicrobial chemicals.…”

“…The phz cluster is organized as an operon of seven genes, the products of which catalyze the synthesis of chorismate and its conversion to PCA (9,27,28,35). This parent compound can be substituted at several positions, depending upon the producer strain, and the genes coding for the enzymes involved in such substitutions are restricted to those strains producing the modified compounds.…”

Substrate specificity of benzamide synthetase involved in 4-hydroxy-3-nitrosobenzamide biosynthesis

“…PhzH catalyzes the amidation of phenazine-1-carboxylic acid to phenazine-1-carboxamide in the biosynthetic pathway of phenazine-1-carboxamide in Pseudomonas aeruginosa PAO1 4 and Pseudomonas chlororaphis PCL1391. 5 OxyD and SsfD are involved in the biosynthesis of oxytetracycline in Streptomyces rimosus 6 and tetracycline SF2575 in Streptomyces sp. SF2575, 7 respectively.…”