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Journal of Bacteriology, March 2001, p. 1909-1920, Vol. 183, No. 6
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.6.1909-1920.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Jesús Mercado-Blanco,1,dagger Koen M. G. M. van der Drift,2,Dagger Per E. Olsson,1 Jane E. Thomas-Oates,2,§ Leendert C. van Loon,1 and Peter A. H. M. Bakker1,*

Department of Plant Ecology and Evolutionary Biology, Section of Plant Pathology, Utrecht University, 3508 TB Utrecht,1 and Department of Mass Spectrometry, Utrecht University, 3584 CA Utrecht,2 The Netherlands

Received 6 July 2000/Accepted 11 December 2000

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.

* Corresponding author. Mailing address: Department of Plant Ecology and Evolutionary Biology, Section of Plant Pathology, Utrecht University, P.O. Box 800.84, 3508 TB Utrecht, The Netherlands. Phone: 31-30-2536861. Fax: 31-30-2518366. E-mail: P.A.H.M.Bakker{at}bio.uu.nl.

dagger Present address: Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible (CSIC), 14080 Córdoba, Spain.

Dagger Present address: Mass Spectrometry Resource, Department of Chemistry, Washington University, St. Louis, MO 63130.

§ Present address: Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester M60 1QD, United Kingdom.

Journal of Bacteriology, March 2001, p. 1909-1920, Vol. 183, No. 6
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.6.1909-1920.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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