Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylicacid and the siderophore pseudomonine in the biocontrol strain Pseudomonasfluorescens WCS374

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, in dicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the bios ynthesis of salicylic acid (SA) and this second siderophore pseudomonine wa s isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis reg ion was identified by transfer of cosmid pMB374-07 to a pseudobactin-defici ent strain of P. putida. Sequence analysis of the 5-kb subclone revealed th e presence of four open reading frames (ORFs). Products of two ORFs (pmsC a nd pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF ( pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also c ontained a putative histidine decarboxylase gene (pmsA). A putative promote r region and two predicted iron boxes were localized upstream of pmsC. We d etermined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promot er. Several lines of evidence indicate that SA and pseudomonine biosynthesi s are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultan eously impaired in SA and pseudomonine production.