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

Abstract

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and 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 contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined 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 promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

“…Duplicated genes indicated by light colors were detected by using a constraint of 35% identity over more than 80% of the length of the protein. for the synthesis of a siderophore related to acinetobactin and containing a salicylamide moiety 21 ( Supplementary Fig. 5).…”

Understanding the involvement of rhizobacteria-mediated induction of systemic resistance in biocontrol of plant diseases

“…Moreover, treatment with strain WCS374 cannot induce systemic resistance in A. thaliana, whereas the application of SA does (Van Wees et al 1997). Possibly, the iron-regulated ISR by WCS374 in radish is mediated by the SA-containing siderophore pseudomonine (Mercado-Blanco et al 2001). Pseudomonine is a nonfluorescent siderophore, produced by WCS374, that was discovered when analyzing nonfluorescent mutants of this strain (Figs.…”

“…2. Structure of pseudomonine, the nonfluorescent siderophore produced by WCS374 (Mercado-Blanco et al 2001). The molecule contains histamine, a cyclothreonine moiety in the middle, and, interestingly, a SA group.…”

Plant growth promoting rhizobacteria and their potential for biocontrol of phytopathogens

“…Optimal suppression of pathogens occurred at levels between 10 -9 -10 -24 M. The critical level of iron at which a siderophoreproducing strain of P. putida suppressed the growth of a fungal pathogen, Fusarium oxysporium, was found to be < 10 -16 M (Neilands and Nakamura, 1991). Since the synthesis of each siderophore generally requires the activity of several gene products (Mercado-Blanco et al, 2001), it is difficult to genetically engineer bacteria to produce modified siderophores. Complementation studies of siderophore-deficient mutants of P. fluorescens M114 indicated that at least five separate genetic loci are needed to encode the enzymes involved in the synthesis of the siderophore pseudobactin M114 (O"Sullivan et al, 1990).…”