Analysis of the pmsCEAB Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain Pseudomonas fluorescens WCS374

摘要

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactinudstill display siderophore activity, indicating the production of a second siderophore. A recombinantudcosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylicudacid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374udwas localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region wasudidentified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysisudof the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsCudand pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein withudstrong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. Theudregion also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and twoudpredicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCRudthat the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genesudwas achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminishedudSA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels ofudSA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicateudthat SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneouslyudimpaired in SA and pseudomonine production.