INTRODUCTION

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Despite its abundance in Earth's crust, iron is largely unavailable for microbial assimilation. Soil microorganisms have developed a mechanism that ensures iron availability in an environment that provides iron (Fe³⁺) at only about 10¹⁸ M. This strategy involves the secretion of low-molecular-weight iron-chelating compounds called siderophores (45) that show a considerable structural diversity and can be classified according to their main chelating groups (26). Bacteria produce a large variety of siderophores under iron-limiting conditions: hydroxamates, phenol-catecholates, and carboxylates. Moreover, some bacteria produce several types of siderophores, as described for strains of Enterobacter cloacae (37) and Pseudomonas spp. (2, 11, 43, 61, 64). Siderophores produced by certain strains of fluorescent Pseudomonas spp. have been linked to suppression of soil-borne plant diseases. It has been suggested that siderophores are antagonistic by means of sequestering iron from the environment, restricting growth of the pathogen (5, 34, 36). Pseudomonas siderophores have also been implicated in inducing systemic resistance (ISR) in plants (33, 40), that is, an enhancement of the defense capacity of the plant against a broad spectrum of pathogens, triggered by nonpathogenic plant growth-promoting rhizobacteria (63).

Another compound with siderophore activity that can be produced by microorganisms is salicylic acid (SA; 2-hydroxybenzoic acid), SA acting as an authentic, endogenous siderophore has been found under iron limitation in Mycobacterium spp. (51), Pseudomonas spp. (2, 3, 43, 64), Azospirillum lipoferum (54), and Burkholderia (Pseudomonas) cepacia (61). SA is also the precursor or intermediate in the biosynthesis of microbial siderophores, such as pyochelin in Pseudomonas aeruginosa (14). Mutants defective in SA production are unable to synthesize pyochelin (2). The salicylamide moiety is encountered not only in pyochelin but also in aeruginoic acid and pseudomonine produced by P. fluorescens strains (3, 12). SA has also been reported to be incorporated into mycobactin S produced by Mycobacterium smegmatis (27, 50), mycobactin T, isolated from M. tuberculosis (60), parabactin produced by Paracoccus denitrificans (47), and maduraferrin isolated from Acinomadura madurae (31). Finally, vulnibactin, a polyamine-containing siderophore from Vibrio vulnificus, contains two SA residues in its molecule (46).

Besides its siderophore activity, bacterial SA has an important role in inducing resistance in plants against pathogen infection. Thus, SA produced by the rhizobacterium P. aeruginosa 7NSK2 has been demonstrated to induce resistance against the fungal pathogen Botrytis cinerea on beans (17, 18). Likewise, heterologous expression of SA biosynthesis genes in P. fluorescens improves ISR in tobacco plants against tobacco necrosis virus (41). In plants, endogenous SA has a key role in systemic acquired resistance, an enhanced resistance status reached by a plant upon pathogen infection (63).

The route of SA biosynthesis in bacteria is not completely known. It has been suggested that in M. smegmatis, salicylate is derived from shikimate (49). Conversion of chorismate to isochorismate and subsequently to salicylate has been obtained from cell extracts of this microorganism (27, 38). Two genes responsible for SA biosynthesis in P. aeruginosa have been identified. The products of pchB and pchA genes have been proposed to catalyze the conversion of chorismate to salicylate via isochorismate (56). P. fluorescens WCS374 is a plant growth-promoting rhizobacterium (PGPR), originally isolated from the rhizosphere of potato (22), that produces SA and the green-fluorescent siderophore pseudobactin at low iron availability (33). Strain WCS374 suppresses Fusarium wilt in radish by ISR, and pseudobactin has been identified as one of the WCS374 traits involved in ISR (33). Likewise, these authors reported that other iron-regulated metabolites induce ISR in bioassays at low iron availability, which might be explained by SA production. However, they also suggested that additional siderophores may play a role in WCS374 ISR induction. The aim of this work was to localize and characterize the SA biosynthesis region of strain WCS374 as well as to identify additional siderophores produced by this strain under low iron availability. During the course of this investigation, evidence was obtained that SA biosynthesis in WCS374 is linked to the synthesis of a siderophore which has been chemically identified as pseudomonine.

	MATERIALS AND METHODS

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Strains, plasmids, and media. The bacterial strains and plasmids used are listed in Table 1. Pseudomonas strains were grown at 28°C in King's medium B (KB) (32), and Escherichia coli strains were grown at 30°C in Luria-Bertani medium (44). SA production was determined from cultures grown in standard succinate medium (SSM; pH 7.0) (42) for Pseudomonas strains and M9 minimal medium (53) for E. coli strains. All plasmids for sequencing were propagated in E. coli DH5 (Clontech) at 37°C. Antibiotics were added at the following concentrations (micrograms per milliliter) when required: ampicillin, 100; kanamycin (KAN), 50; nalidixic acid (NAL), 25; and tetracycline (TET), 20 (for E. coli) and 40 (for Pseudomonas).

DNA manipulations. Standard procedures were used for DNA electrophoresis, DNA transfer from agarose gels to nylon membranes (Boehringer Mannheim Biochemicals), preparation of competent cells, and transformation (53). Small-scale isolation of recombinant plasmids was done by alkaline lysis as described by Birnboim and Doly (9). Purification of plasmid DNA and elution of DNA restriction fragments from agarose gels were performed using Qiagen Inc. systems according to the instructions provided. DNA restrictions and ligations with T4 DNA ligase were conducted with enzymes purchased from Pharmacia according to the manufacturer's instructions. Hybridization was done with a nonradioactive detection kit from Boehringer Mannheim Biochemicals, and the chemiluminescence method was used to detect hybridization bands.

Tn5 mutagenesis and bacterial matings. To perform random mutagenesis of P. fluorescens WCS374, the mobilization system of strain S17-1 and the suicide vector pSUP2011, carrying transposon Tn5, were used (59). Matings were carried out by mixing overnight cultures of donor strain S17-1 harboring pSUP2011 and recipient strain WCS374 in a ratio 1:1. After pelleting the cultures, the mating mixture was resuspended in a small volume (50 to 100 µl) and loaded onto a sterile Millipore filter (0.45-µm pore size) on KB agar plates. After 24 h of incubation at 28°C, cells were resuspended in fresh KB, and aliquots were plated on KB agar plates supplemented with KAN and NAL, to which WCS374 is intrinsically resistant. To avoid counterselection of mutants affected in iron uptake and metabolism, 100 µM FeCl₃ · 6H₂O was added to the selection medium. The transposition frequency in these experiments was 10⁵ per recipient cell. Km^r colonies were then screened for lack of fluorescence under UV irradiation on KB agar plates, growth on KB agar plates amended with 800 µM 2,2-bipyridyl (BDH) (which acts as an iron chelator) (39), growth on SSM agar plates, and inability to produce orange halos on chrome azurol S (CAS) medium agar plates (55). The presence of single transposition events in siderophore-deficient mutants was checked by hybridization. Total DNA from the Km^r mutants was isolated, digested with EcoRI, blotted onto a membrane, and hybridized against the digoxigenin-labeled internal HindIII fragment of Tn5, using high-stringency conditions.

Siderophore determination. Siderophore production by bacterial strains was detected by using the universal siderophore detection medium CAS agar (55). A siderophore producer colony chelates iron from the medium, and a shift from blue (chelated CAS) to orange (unchelated CAS) is obtained. Droplets (3 µl) of an overnight SSM culture were spotted on plates, and after incubation (1 to 3 days, at 28 or 37°C) the relative halo size ([halo diameter  colony diameter]/halo diameter]) was determined. Halo determination was done in triplicate and repeated at least three times.

(i) In vitro SA production. SA production from culture supernatants was determined as previously described (33, 43). Strains of Pseudomonas spp. were grown in liquid SSM at 28°C; E. coli strains were grown in M9 medium at 30°C. After removal of cells by centrifugation, culture supernatants were acidified with 1 N HCl to pH 2, and SA was extracted into CHCl₃ upon vigorous shaking (culture supernatant:CHCl₃, 1:1). For low levels of SA production, 1 volume of CHCl₃ was used to extract SA from up to 3 volumes of spent medium. An absorbance spectrum (200 to 400 nm) of the organic phase was determined to obtain a qualitative determination. For quantitative measurements, 1 volume of H₂O and 1.25 × 10³ volumes of 2 M FeCl₃ · 6H₂O were added to the CHCl₃ phase. The absorbance of the purple iron-SA complex developed in the aqueous phase was measured at 527 nm, using SA dissolved in the growth medium and treated as described above as a standard.

(ii) Purification and identification of the siderophore pseudomonine. A lyophilized culture filtrate of P. fluorescens WCS374-08, grown in liquid SSM, was redissolved in water to a concentration of 100 mg/ml; 1 ml of this solution was applied to an Alltech C18 reversed-phase solid extraction column (5 ml), conditioned by washing with 5 ml of 100% acetonitrile (ACN), 5 ml of 40% ACN, and 5 ml of H₂O. After the sample solution was applied, the column was washed four times with 5 ml of H₂O. The pseudomonine was eluted with 1 ml of 40% ACN followed by 1 ml of 100% ACN. Both fractions were shown to contain pseudomonine, and both were lyophilized. The pseudomonine eluted in 100% ACN was redissolved in 50 µl of 5% acetic acid (AcOH), and 2 µl was loaded into the glycerol matrix for fast atom bombardment (FAB) mass spectroscopy and collision-induced dissociation (CID) analysis. The pseudomonine eluted in 40% aqueous ACN was redissolved in 50 µl of ethanol, and approxiamtely 10 µl was analyzed by probe electron ionization (EI). For H-D exchange, the fraction was redissolved in 100 µl 5% deuterated AcOH, and 2 µl was loaded into a D-glycerol matrix.

1

Construction of a gene library and cloning of SA biosynthesis genes. Genomic WCS374 DNA was isolated and partially digested with EcoRI. Sucrose gradient fractionation, agarose gel electrophoresis for size analysis, fragment DNA preparation, and ligation reaction with EcoRI-digested cosmid vector pLAFR1 were performed as described by Sambrook et al. (53). In vitro packaging and transduction of the ligated DNA were performed as instructed by the manufacturer (Boehringer Mannheim Biochemicals).

Deletion analysis of the SA biosynthesis region and DNA sequencing. The region involved in SA biosynthesis in P. fluorescens WCS374 was mapped by subcloning and deletion analysis of plasmid pMB374-07 (Fig. 1). The 5-kb EcoRI fragment from pMB374-07 containing the SA biosynthesis region was subcloned in pGEM-3Z (Promega) in both orientations with respect to the lacZ promoter, yielding plasmids pE3 and pE3R. Plasmid DNAs were digested using the single XbaI and SmaI sites present in the multiple cloning site of pGEM-3Z. XbaI was used to generate 5' protruding ends which were protected from exonuclease III digestion with -phosphorothioate-derived deoxynucleoside triphosphates (dNTPs) plus Klenow enzyme. Subsequently, SmaI was used to generate blunt ends susceptible to exonuclease III digestion. Templates were generated by progressive unidirectional deletions using the Erase-a-Base system from Promega Corporation according to the manufacturer's instructions. The resulting fragments were ligated and transformed into DH5 cells. Plasmids from ampicillin-resistant transformants with suitable deletions were selected, purified using a Qiagen kit, and used for DNA sequencing. Selected deletion plasmids were screened for the ability to direct SA biosynthesis in E. coli as described above.

View larger version (19K): [in this window] [in a new window]   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.

Use of RT-PCR to study pms gene expression. Expression of pms genes in cultures under low- and high-iron conditions was studied in reverse transcriptase-mediated PCR (RT-PCR) experiments. We used mutant 374-08 as an internal gene expression control since it contains the neomycin phosphotransferase gene (neo) (7). This gene, carried by the Tn5 element present in this mutant, is expressed when KAN is present in the incubation medium, and its expression can be compared to that of the pms genes. Total RNA was isolated from strains WCS358 (used as a negative control for SA production), WCS374, and 374-08 with a Qiagen RNeasy kit according to the manufacturer's instructions. Bacteria were grown in liquid SSM (15 ml) for at least 48 h at 28°C. For high-iron conditions, cells from 48-h-old cultures were spun down and resuspended in 15-ml of fresh SSM, amended with 100 µM FeCl_{3 ·} 6H₂O. The cells were then incubated for 3 h at 28°C; 10⁹ cells from low- and high-iron cultures were used for total RNA isolation. RNA was eluted in 50 µl of pyrocarbonic acid diethyl ester (DEPC)-treated water. RNA concentration and quality were determined spectrophotometrically and by gel electrophoresis, respectively. To exclude the possibility of contamination with traces of DNA, RNA samples were treated with 20 U of RNase-free DNase (Pharmacia Biotech) for 30 min at 37°C. RNAs were subsequently purified by the method described in the RNeasy kit for RNA cleanup. Simultaneously, DNA samples of strain WCS374 and mutant 374-08 (2 µg of genomic DNA in 50 µl of DEPC-treated water) were treated with RNase-free DNase, as controls to check the effectiveness of the DNase treatment.

Nucleotide sequence accession number. The nucleotide sequence of the P. fluorescens WCS374 pmsCEAB genes has been deposited in the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession number Y09356.

	RESULTS

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P. fluorescens WCS374 produces three siderophores at low iron availability. To obtain mutants impaired in siderophore production, random Tn5 mutagenesis of P. fluorescens strain WCS374 was carried out. Upon screening of some 4,000 Km^r colonies, 13 nonfluorescent (deficient in production of pseudobactin [Psb-deficient]) mutants on KB agar plates were isolated. However, all of the mutants were still able to grow on KB supplemented with 2,2-bipyridyl. This result could be explained either by the production of SA, which can act as a siderophore, or by the presence of third siderophore other than pseudobactin and SA in strain WCS374.

View this table: [in this window] [in a new window]   TABLE 2.   SA production in SSM by different Pseudomonas strains (assay A) and in M9 medium by E. coli DH5 harboring different plasmids constructed in this work (assay B)

Identification of a mutant impaired in production of both SA and a newly detected siderophore and construction of SA Psb-deficient mutants. During the screening for Psb-deficient mutants, a mutant (374-05) showing enhanced production of pseudobactin compared to that by P. fluorescens WCS374 was also isolated. When analyzed for SA production, the result was negative (Table 2). The results obtained in the CAS medium agar assay revealed that the halo produced at 28°C was not significantly different from the one produced by WCS374 or the Psb-deficient mutants. However, incubation at 37°C showed a strong reduction of the relative halo size, due to repression of pseudobactin synthesis at this temperature (39). Actually, pseudobactin was masking the detection of the additional siderophore in CAS agar plates. In contrast, no reduction in halo size at 37°C was observed for the Psb-deficient mutant 374-08. This observation indicated that mutant 374-05 was impaired in the production of not only SA but also the unidentified siderophore. Yet, siderophore activity was detectable in mutant 374-05 at 37°C.

Identification of the additional siderophore as the SA-based siderophore pseudomonine. Attempts to identify pyochelin, an SA-based siderophore, in culture supernatants of the strains tested failed. Moreover, neither catechol nor hydroxamate groups were detected in the supernatant of these strains. In contrast, weak positive reactions for both were obtained in pseudobactin producer strains WCS374 and 374-05. This can be explained by the presence of these groups in the structure of this siderophore (26). To identify the siderophore produced by WCS374, culture supernatants of mutant strain 374-08 grown in liquid SSM were used.

(i) Mass spectrometric analysis of pseudomonine. The siderophore preparation was analyzed in the positive-ion mode using FAB mass spectrometry and CID, as well as EI. In both the positive-ion mode FAB mass spectrum recorded from the siderophore isolated in this study and the (unpublished) positive-ion mode FAB mass spectrum recorded by Anthoni and coworkers from pseudomonine (3), a protonated molecule was observed at m/z 331. To obtain structural information, the ion at m/z 331 was submitted to CID tandem mass spectrometry (Fig. 2A). The most abundant fragment ion is observed at m/z 211 and corresponds to the loss of the SA group (Fig. 2B). The SA group fragment is observed at m/z 121. The ion at m/z 110 corresponds to a histidine immonium ion, which is derived from the histamine moiety, while the ions at m/z 82 and 95 also arise by cleavage of the hydrocarbon backbone of the histamine moiety. The ion at m/z 138 corresponds to cleavage in the cyclothreonine moiety with charge retention on the histamine-containing part of molecule. We assign the ion at m/z 204 as deriving from the SA-containing portion of the molecule and being generated by cleavage in the cyclothreonine moiety. This ion is complemented by an ion at m/z 128 that we assign as arising via an analogous cleavage in the cyclothreonine moiety, but now with charge retention on the histamine-containing part of the molecule.

View larger version (16K): [in this window] [in a new window]   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.

(ii) NMR analysis of pseudomonine. To obtain further structural information from the bioactive compound isolated, the fraction containing the bioactive compound was analyzed by ¹³C and ¹H NMR. The chemical shifts are summarized in Table 3, alongside the NMR data obtained by Anthoni et al. (3). A comparison of the two data sets makes it clear that the chemical shifts observed by both groups are nearly identical.

SA and pseudomonine biosynthesis loci are linked in the strain WCS374 genome: delimitation of the SA biosynthesis region. To localize a recombinant cosmid clone harboring the biosynthetic regions of SA and pseudomonine, heterologous expression was pursued. JM218, a Tn5 Psb-deficient mutant of P. putida WCS358 (39), produces a very small halo on CAS medium agar plates. Both WCS358 (33) and JM218 (Table 2) do not produce SA. Thus, halo restoration could be used as a marker for expression of WCS374 siderophore biosynthetic genes in JM218. Matings were performed using a mixture of the gene library (>8,000 clones; average insert size, 20 kb) in DH5 as donor strain and JM218 as recipient strain. Helper plasmid pRK2013 (20) was used to aid transfer of the recombinant cosmids. Selection of the transconjugants was made on KB without Mg^{2 +} (to avoid antagonism with TET), supplemented with TET, KAN, and NAL. After screening 2,150 Tc^r and Km^r JM218 transconjugants, we isolated 36 clones capable of inducing halo production in CAS medium agar plates. Only those clones that showed halos not only at 28°C but also at 37°C, where pseudobactin synthesis is repressed (39), were selected. The three clones that fulfilled this criterion (pMB374-07, pMB374-15, and pMB374-31) were also the only ones showing SA production. However, the amount of SA detected was far lower than that obtained for WCS374 (33) (Table 2). SSM culture supernatants of JM218 carrying these recombinant clones showed a blue fluorescence under UV light, which corroborates the observation obtained for the Psb-deficient mutants of WCS374. High-pressure liquid chromatography (HPLC) analysis of the culture supernatant of JM218 carrying pMB374-07 revealed the presence of pseudomonine (data not shown).

Nucleotide sequence analysis of the SA biosynthesis region. Nucleotide sequence analysis of the 5,057-bp EcoRI fragment (56.48% G+C content) revealed the presence of four major open reading frames (ORFs) with high coding probability and rightward polarity (Fig. 1). The first ORF (ORFA) is preceded by a strong potential ribosome binding sequence (RBS) (GGAGG) that matches the consensus RBS of E. coli (58).

Protein sequence homology. A search in the databases for protein homology yielded relevant data for all predicted translational products of the sequenced region. ORFA (proposed name pmsC, for pseudomonine synthesis) codes for a protein with a predicted molecular weight of 43,102. The deduced amino acid sequence showed similarities with several chorismate-utilizing enzymes: isochorismate synthases (ICSs) of E. coli (EntC; EMBL/Swiss-Prot accession number P10377; 45% identity and 73% similarity), Aeromonas hydrophila (AmoA; P23300; 43% identity and 69% similarity), Vibrio cholerae (VibC; 007898; 43% identity and 69% similarity), Bacillus subtilis (DhbC; P45744; 40% identity and 71% similarity), and P. aeruginosa (PchA; Q51508; 38.5% identity and 52.6% similarity in 192 residues of the carboxy-terminal domain). It also showed moderate homology with menaquinone-specific ICS (MenF) from different bacteria and weak homology with component I of anthranilate synthases (TrpE) and p-aminobenzoate synthase component I (PabB) from different organisms. ICS catalyzes the conversion of chorismate to isochorismate. This compound is utilized for the biosynthesis of some quinones and phenolic acid derivatives such as 2,3-dihydroxybenzoic acid (2,3-DHB). ICSs cited above have been reported as key enzymes in the biosynthetic pathway of siderophores such as enterobactin (19), pyochelin and SA (56), amonabactin (6), and vibriobactin (O07898).

Expression of SA genes in E. coli. SA was detected in M9 culture supernatants of E. coli DH5 cells carrying plasmid pE3 or pE3R (Table 2), suggesting that the putative promoter detected upstream of pmsC is functional in E. coli. Although high SA levels were detected, irrespective of orientation, enhanced SA production was obtained when the genes were properly oriented with regard to the lacZ promoter present in pGEM-3Z (pE3R). SA was also detectable in the spent medium of DH5 cells harboring different deletion clones, as well as subclones (Table 2). Deletions affecting pmsB (plasmid pE3-01) (Fig. 1) failed to produce SA in DH5, indicating that this gene is essential for SA biosynthesis. Deletions affecting genes upstream of pmsB (i.e., plasmid pE3R-01 [Fig. 1], a 233-bp deletion derivative affecting the potential promoter region and the first nine codons of pmsC) resulted in 10-fold-lower SA production (Table 2). Likewise, DH5 carrying plasmids pJMSal-10 and pJMSal-20 produced detectable amounts of SA (Table 2).

Expression of pms genes is iron regulated. DNA sequence analysis identified putative ⁷⁰ promoter sequences only upstream of pmsC, suggesting that pms genes might have a polycistronic organization. The presence of two potential Fur boxes suggests that expression of the pms genes is iron regulated. Expression of pms genes was analyzed by RT-PCR. Total RNA from strains WCS374 and 374-08 grown in SSM was used for cDNA synthesis using primer SAL03. For subsequent PCR experiments, we used several pairs of primers that overlapped different pms genes: SAL01-SAL02 to define an internal pmsB PCR product, HDC01-SAL02 to yield a PCR product spanning pmsAB, and DHB01-DHB02 to amplify a fragment that partially overlaps the pmsCE coding region (Fig. 3A). For cDNA synthesis from neo mRNA, primer KM03 was used. The internal primer pair KM01-KM02 was used for PCR. If a polycistronic mRNA is obtained from the cDNA synthesis using primer SAL03, the predicted PCR products will be detected in all primer combinations designed. The result of this experiment is shown in Fig. 3B. PCR products expected for SAL01-SAL02 (214 bp), HDC01-SAL02 (599 bp), and DHB01-DHB02 (439 bp) were detected in mutant 374-08 and WCS374 cDNA PCRs. Similarly, the PCR product predicted for KM01-KM02 (405 bp) was detected in mutant 374-08 cDNA but not in WCS374 cDNA. The band sizes were concordant with PCR products obtained in PCR control reactions using WCS374 and 384-08 genomic DNAs as templates. Simultaneously, control PCRs of genomic DNA treated with RNase-free DNase did not yield any product, indicating that PCR bands due to DNA contamination in RNA samples can be excluded.

View larger version (75K): [in this window] [in a new window]   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.

	DISCUSSION

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Several strains of Pseudomonas spp. have been reported to produce more than one type of siderophore under iron-limiting conditions (2, 11, 43, 64). For the PGPR strain P. fluorescens WCS374, production of an additional siderophore, besides pseudobactin and SA, was suggested (33). In this work, we show that the additional siderophore in strain WCS374 is pseudomonine, an isoxazolidone previously isolated in culture supernatants of iron-deficient cultures of a P. fluorescens strain isolated on spoiled Nile perch from Lake Victoria (Africa) (3).

Two lines of evidence suggest that SA production and pseudomonine synthesis are related. One mutant that we studied (374-05) is impaired in both SA and pseudomonine production, and a recombinant cosmid from the WCS374 gene library carries the genetic information for both traits. Additionally, pseudomonine has been identified as an SA-based siderophore (reference 3 and this work). SA and pseudomonine biosynthesis genes are closely linked and present in the recombinant clone pMB374-07. This plasmid induced both siderophore activity and SA production in P. putida WCS358 Psb-deficient strain JM218. pMB374-07 derivative subclones were unable to drive pseudomonine synthesis, suggesting that most of the cloned insert is involved in this trait. However, only 5 kb from pMB374-07 were required to induce SA production in JM218 (plasmids p07E3 and p07E3R) and in E. coli cells (plasmids pE3 and pE3R).

Double mutants impaired in the production of both pseudobactin and pseudomonine still showed some siderophore activity in CAS medium. Although additional iron uptake systems could still be functional, the possibility that mutant 374-05 is leaky and able to release small quantities of pseudomonine to the medium cannot be ruled out. As a matter of fact, HPLC analysis of the culture supernatant of mutant 374-05.8 revealed a very small peak at the position of pseudomonine (data not shown), indicating that the mutant indeed is leaky. Preliminary experiments have shown that the Tn5 insertion in mutant 374-05 is located elsewhere in the WCS374 genome and that this mutant is likely affected in pseudomonine transport machinery (J. Mercado-Blanco, unpublished data).

Serino et al. (56) proposed that SA synthesis in P. aeruginosa proceeds from chorismate via isochorismate. The two potential SA biosynthesis genes of strain WCS374, pmsC and pmsB, showed moderate to high similarity to the SA biosynthetic genes pchB and pchA of P. aeruginosa (56); PchA would act as an ICS, and PchB would act as an isochorismate-pyruvate lyase. Based on sequence similarities, pmsC and pmsB may function similarly in P. fluorescens WCS374. However, the genetic organization is different. In P. aeruginosa, pchB and pchA are the distal genes of the pchDCBA operon involved in SA and pyochelin biosynthesis (57). In P. fluorescens WCS374, two genes are interspersed between the proposed SA biosynthetic genes (pmsC and pmsB): a putative entE homolog (pmsE) and a putative hdc gene (pmsA). It is unlikely that there is any role in SA synthesis for these latter genes, especially pmsA with a totally unrelated activity. We speculated that this genetic arrangement is part of the biosynthetic machinery of pseudomonine. pmsC and pmsB would contribute to synthesis of the SA moiety present in the pseudomonine structure. The presence of an hdc gene (pmsA) supports the conclusion that histamine, which is present in the structure of pseudomonine, is a precursor in the synthesis of this siderophore. Anguibactin in V. anguillarum (62) and acinetobactin in A. baumannii (65) are siderophores that also contain this biogenic amine. Acinetobactin and pseudomonine possess an oxazoline ring instead of a thiazoline ring which is present in anguibactin. Finally, PmsE has striking similarities to proteins involved in the biosynthesis of siderophores such as enterobactin (EntE), pyochelin (PchD), vibriobactin (VibE), and yersiniabactin (YbtE). The EntE protein mediates the activation of a catechol (2,3-DHB) in one of the steps in enterobactin synthesis. As proposed by Serino et al. (57) and previously reported for EntE of E. coli (35), PmsE might act in the same way as EntE, activating the carboxylate group of SA.

In vivo SA biosynthesis was achieved in heterologous backgrounds such as in P. putida and E. coli cells (Table 2). Similarly, pms genes were expressed under the control of their own promoter in E. coli (Table 2). Only when ORFD (pmsB) was not present was SA production impaired. When pmsB was provided alone (plasmid pJMSa1-10), SA was still detectable in M9 medium supernatant (Table 2). Thus, pmsB has been shown to be essential for SA synthesis in E. coli. This observation indicates that PmsB can use endogenous substrates provided by E. coli cells. However, upstream genes appear to provide additional substrates to produce high SA levels (plasmids pE3 and pE3R [Table 2]). Serino et al. (56) concluded that PchB in P. aeruginosa seems to be involved in the conversion of isochorismate to SA, but it is unable to synthesize SA directly from chorismate. The fact that SA was detected in DH5 harboring deletion plasmids affecting pmsC indicates that this gene is not essential for SA biosynthesis in E. coli but is important to reach the SA levels produced by pE3R. An explanation may be that indigenous ICS activity complements the absence of PmsC in deletion clones. Alternatively, substrates provided by E. coli can be used, with less specificity, by PmsB. In contrast, SA was not detected in JM218 culture supernatants when pmsB was provided alone (pLASa1-10). PmsB has a certain degree of homology with the N-terminal domain of different chorismate mutases. An interesting possibility, which require further experimental support, is that the putative PmsB can use other substrates, including chorismate. In this sense, moderate homology (29% identity and 59% similarity) was found with the 129-amino-acid-residue protein PapB from Streptomyces pristinaespiralis, a protein identified as a mutase involved in the biosynthesis of 4-methylamino-L-phenylalanine (accession number P72541). It is also interesting that PmsB showed homology with an unknown ORF in V. vulnificus. This bacterium synthesizes vulnibactin, a siderophore which contains two residues of SA (46). These two residues are involved in the formation of oxazoline rings with L-threonine, the same arrangement as encountered in pseudomonine. Thus, PmsB, PchB, and the latter ORF may constitute a family of proteins involved in synthesis of the SA moiety in this type of siderophore.

Results of homology analysis presented in this work, along with the results of gene expression by RT-PCR as well as Fe-mediated SA synthesis repression studies performed earlier by our group (33), show that (i) the promoter region detected upstream of pmsC is functional, (ii) expression of the pms genes is iron regulated, and (iii) the pmsCEAB transcript is polycistronic. However, the existence of additional promoters in the pms region cannot be completely ruled out. Differences in efficiency in primer binding can be considered, although no obvious differences were observed when DNA templates were used in PCR control experiments (Fig. 3B).

The specific contributions of pseudobactin, pseudomonine, and SA in iron uptake in strain WCS374 are not known. On the other hand, it has been demonstrated that pseudobactin and SA induce resistance in radishes against Fusarium wilt (33). These authors reported that a pseudobactin mutant was still able to induce resistance at low iron availability, hypothesizing that SA or a different siderophore, which now has been confirmed to be pseudomonine, was responsible for disease suppression. The possible role of pseudomonine in plant disease suppression and ISR will be further investigated.