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.

FIG. 1

(A) Restriction map of the 28-kb insert cloned in plasmid pMB374-07. The two lines above the restriction map represent subclones which showed the phenotypes indicated (SA⁻ and/or Pms⁻) when harbored by JM218. (B) Physical map of the minimal 5-kb EcoRI fragment responsible for SA biosynthesis. Lines below the restriction map represent different deletion clones and subclones constructed to delimit the minimal region involved in SA biosynthesis in E. coli. Production [(+)] or nonproduction [(−)] of SA is indicated. Restriction sites: E, EcoRI; B, BamHI; H, HindIII; S, SalI; Sc, SacI; Pv, PvuII; RV, EcoRV.

FIG. 2

(A) CID mass spectrum of the compound found in the fraction eluting in 100% ACN. (B) Fragmentation scheme for the component with [M + H]⁺ at m/z 331. Boldface numbers indicate fragment masses of the ions obtained from the deuterated component. Small numbers refer to the atom numbering used to assign the NMR data summarized in Table 3. The H atoms shown in boldface correspond to exchangeable protons.

FIG. 3

Analysis of pms gene expression by RT-PCR. (A) Scheme representing positions of the primers used in this study, as well as lengths of the expected PCR products. Restriction sites are abbreviated as in Fig. 1. (B) RT-PCR products obtained from cDNA samples obtained after total RNA isolation of bacterial cultures grown under low- and high (Fe³⁺)-iron conditions. Experiments were performed as indicated in the Materials and Methods. One microliter of cDNA mixture was used in each reaction in lanes 1 to 18; 1 μl (2 ng) of DNA was used in control reactions (lanes 19 to 23). Lanes: M, size markers; 1, WCS358 (primer pair SAL01-SAL02); 2, WCS358 (Fe³⁺) (SAL01-SAL02); 3, WCS358(KM01/KM02); 4, WCS358 (Fe³⁺) (KM01-KM02); 5, WCS374 (SAL01-SAL02); 6, WCS374 (Fe³⁺) (SAL01-SAL02); 7, WCS374 (KM01-KM02); 8, WCS374 (Fe³⁺) (KM01-KM02); 9, 374-08 (SAL01-SAL02); 10, 374-08 (Fe³⁺) (SAL01-SAL02); 11, 374-08 (HDC01-SAL02); 12, 374-08 (Fe³⁺) (HDC01-SAL02); 13, 374-08 (DHB01-DHB02); 14, 374-08 (Fe³⁺) (DHB01-DHB02); 15, 374-08 (KM01-KM02); 16, 374-08 (Fe³⁺) (KM01-KM02); 17, WCS374 (DHB01-DHB02); 18, WCS374 (Fe³⁺) (DHB01-DHB02); 19, 374-08 DNA (SAL01-SAL02); 20, 374-08 DNA (HDC01-SAL02); 21, 374-08 DNA (DHB01-DHB02); 22, 374-08 DNA (KM01-KM02); 23, WCS374 DNA (KM01-KM02); 24, WCS374 DNA (2 μg) treated with RNase-free DNase (SAL01-SAL02); 25, 374-08 DNA (2 μg) treated with RNase-free DNase (KM01-KM02). (C) Dilution series of cDNA samples (1 μl) used in PCR experiments to estimate repression level upon Fe³⁺ addition. Primers used were SAL01-SAL02 for pmsB expression (214-bp PCR band) and KM01-KM02 for neo expression (405-bp PCR band). All lanes correspond to cDNA obtained from total RNA of 374-08. Odd-numbered lanes correspond to minimal-iron conditions; even-numbered lanes to 100 μM Fe³⁺. Lanes 1 to 4, 1 μl of the cDNA original mixture; lanes 5 to 8, 5-fold-diluted samples; lanes 9 to 12, 25-fold-diluted samples; lanes 13 to 16, 125-fold-diluted samples; lanes 17 to 20, 625-fold-diluted samples; lanes 21 to 24, 3,125-fold-diluted samples.