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Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, Campus Sur, Santiago de Compostela 15782, Spain
Correspondence
Manuel L. Lemos
mlemos{at}usc.es
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence reported in this paper is AM168450.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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).
The fish pathogen Vibrio anguillarum is the causative agent of vibriosis, a highly fatal haemorrhagic septicaemia affecting marine and freshwater fish species throughout the world (Toranzo & Barja, 1990). Up to 23 serotypes, designated O1–O23, are recognized in the European system (Pedersen et al., 1999), with O1 and O2 being the main ones implicated in disease (Toranzo et al., 1997). Although the virulence mechanisms of V. anguillarum are not fully understood, it is known that the ability to scavenge iron through the utilization of siderophores contributes significantly to virulence (Wolf & Crosa, 1986). Two clearly different siderophore-mediated systems have been described in V. anguillarum strains belonging to serotypes O1 and O2 (Conchas et al., 1991). In most pathogenic strains of serotype O1, the system is mediated by the 65 kb plasmid pJM1, which harbours genes for the biosynthetic machinery, and utilization of the catecholate-type siderophore anguibactin (Stork et al., 2002). Recently, it has been reported that the biosynthesis of anguibactin needs chromosomally encoded enzymes, in addition to those encoded by pJM1 genes (Alice et al., 2005).
On the other hand, all serotype O2 strains tested so far, and some plasmidless serotype O1 strains, produce a siderophore that is encoded by hitherto uncharacterized chromosomal genes, and is not related to the pJM1 plasmid-mediated system (Stork et al., 2002). The biological activity deduced from cross-feeding assays pointed initially to a functionality related to the Escherichia coli 2,3-dihydroxybenzoic acid (DHBA)-containing siderophore enterobactin (Lemos et al., 1988; Conchas et al., 1991). This chromosome-encoded system produces a siderophore called vanchrobactin, which has been recently isolated from the serotype O2 strain RV22, and chemically characterized as N-[N'-(2,3-dihydroxybenzoyl)-arginyl]-serine (Soengas et al., 2006).
The genes necessary for DHBA biosynthesis are also present and expressed in the chromosome of the anguibactin-producing V. anguillarum O1 strain 775 (Chen et al., 1994); in addition, a chromosomal gene cluster involved in DHBA synthesis and activation has been identified in this strain (Alice et al., 2005). Most of these genes have functional homologues in the pJM1 plasmid, except angAch (encoding a putative 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase), which is essential for DHBA and anguibactin synthesis, and for which only the chromosomal copy seems to be functional.
Since vanchrobactin is a catechol siderophore (Lemos et al., 1988; Soengas et al., 2006), we hypothesized that homologues of the genes described in the chromosome of serotype O1 strains, and some additional genes, could be responsible for biosynthesis of DHBA and the siderophore produced by plasmidless V. anguillarum strains. Thus, the aim of this study was the identification and characterization of the genes involved in the biosynthesis of vanchrobactin.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. V. anguillarum serotype O2 strain RV22 was routinely grown at 25 °C in tryptic soy agar and broth (Difco), supplemented with 1 % NaCl (TSA-1 and TSB-1, respectively). E. coli strains were routinely grown at 37 °C in Luria–Bertani (LB) medium (Pronadisa) supplemented with the appropriate antibiotics. All strains were stored frozen at –80 °C in LB broth containing 20 % (v/v) glycerol. Ampicillin sodium salt and kanamycin (Sigma-Aldrich) stock solutions in ultrapure water (100 mg ml–1 and 50 mg ml–1, respectively) were filter-sterilized, and stored at –20 °C.
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. Total genomic DNA from V. anguillarum was purified with the Easy-DNA kit (Invitrogen). Plasmid DNA purification, and extraction of DNA from agarose gels, were carried out using kits from Qiagen. DNA-probe labelling and Southern blot analysis were performed with the ECL DNA-labelling and detection system (Amersham Biosciences). PCR reactions were carried out in a T-Gradient Thermal Cycler (Biometra), with Taq polymerase BioTaq (Bioline). For inverse PCR, chromosomal DNA was digested with a single restriction enzyme, self-ligated, and used as template in a PCR reaction with the Expand Long Template PCR System (Roche Diagnostics), using suitable primers.
DNA sequences were determined with the dideoxy-chain-termination method on either plasmid or PCR products using GenomeLab DTCS-Quick Start Kit with a CEQ 8000 DNA Sequencer (Beckman Coulter). Sequences were examined and assembled using BioEdit version 7.0.4.1 (Hall, 1999). The European Bioinformatics Institute and the NCBI services were used to consult the DNA and protein sequence databases with FASTA3 and BLAST algorithms. The protein families database of alignments and hidden Markov models (Pfam), of the Sanger Institute, was utilized to predict the protein domain organization (Bateman et al., 2004). The prediction of transmembrane helices in proteins was carried out using program HMMTOP version 2.0 (Tusnady & Simon, 2001).
Cloning and sequencing of a gene cluster involved in vanchrobactin biosynthesis.
In order to clone the genes responsible for the biosynthesis of vanchrobactin, in the chromosome of serotype O2 strain RV22, we used PCR to look for the presence of a homologue of the angE gene originally cloned from the chromosome of a pJM1-like plasmid-bearing V. anguillarum O1 strain 90-11-287 (Holmstrom & Gram, 2003). As a result, we found an ORF that showed 97 % identity to the nucleotide sequence of this angE gene. An internal fragment of the angE homologue amplified in RV22 was labelled, and used as a probe in Southern hybridizations to identify suitable restriction sites surrounding this locus. This information was used to carry out an inverse PCR reaction, and a 7 kb fragment was obtained. Successive inverse PCRs and primer walking allowed us to extend sequencing in two directions to a fragment of approximately 18 kb.
Mutant construction.
Gene deletions in V. anguillarum RV22 were constructed by using PCR amplifications of two fragments of each gene and flanking regions, which when ligated together would result in an in-frame (nonpolar) deletion (Fig. 1). The oligonucleotides used to amplify the upstream and downstream ends of each gene are listed in Table 2. Suitable restriction sites were added to the oligonucleotides. Construction of in-frame deletions of vabA, vabC, vabE, vabB, vabS and vabF was accomplished in several steps. The PCR-amplified 3'-end gene fragments were ligated into pWKS30, and resulting plasmids were cut with suitable enzymes, and ligated to the 5' ends of the PCR fragments of each gene. Each deleted allele cloned in pWKS30 was digested with NotI and ApaI, and ligated into the suicide vector pNidKan (Mouriño et al., 2004). As a pCVD442 derivative, pNidKan contains R6K ori, requiring the pir gene product for replication, and the sacB gene, conferring sucrose sensitivity. The resulting plasmids were mated from E. coli S17-1-
pir into V. anguillarum RV22, and transformants, with the plasmid integrated in the chromosome by homologous recombination, were selected on agar medium containing 50 µg kanamycin ml–1 (resistance conferred by pNidKan) and 50 µg ampicillin ml–1 (specific antibiotic to select V. anguillarum RV22). A second recombination event was obtained by selecting for sucrose (10 %) resistance. This process led to the production of the following mutant strains of V. anguillarum: MB12 (
vabA), MB10 (vabC), MB3 (vabE), MB11 (vabB), MB6 (vabS), MB14 (vabF) and MB19 (vabH). Deletion of the parental gene was verified in all cases by Southern blot hybridization. DNA sequencing of the region involved in the deletion was carried out to ensure that all constructs were in-frame.
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). The vabF gene was amplified from strain MB19 (vabH) using proFesV_XbaI and VacF_XbaI primers (Table 2) to incorporate the promoter upstream of vabH. The amplified DNA fragments were cloned into the pHRP309 vector (Parales & Harwood, 1993) at the appropriate restriction site, and subsequently transformed into the E. coli S17-1-pir strain. The resulting plasmids (Table 1) were mated from E. coli S17-1-pir into the corresponding V. anguillarum mutant, and transformants were selected on agar medium containing 10 µg gentamicin ml–1 (resistance conferred by pHRP309) and 50 µg ampicillin ml–1 (to select for V. anguillarum). The pHRP309 recombinant plasmids were isolated from the V. anguillarum mutants, and digested with appropriate restriction enzymes to verify the presence of the inserts.
Growth under iron-limited conditions, and test for siderophore production.
To test the ability of V. anguillarum defective mutants to grow under iron-limited conditions, overnight cultures in LB of the parental and mutant strains were adjusted to an OD600 of 0.5, and diluted 1 : 15 in CM9 minimal medium (Lemos et al., 1988) containing the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA) at 5 µM. Cultures were incubated at 25 °C, with shaking at 150 r.p.m., and growth (OD600) and siderophore production were measured after 22 h incubation. Siderophore production was measured using the chrome azurol-S (CAS) liquid assay (Schwyn & Neilands, 1987). In addition, the Arnow test (Arnow, 1937) was used for spectrophotometric measurement of catechol in each sample. A non-inoculated CM9 sample, containing EDDA at an appropriate concentration, was used as negative control and spectrophotometric blank for the CAS liquid assay.
Cross-feeding assays.
The biological activities of the supernatants produced by the mutant strains were determined by performing cross-feeding experiments. We tested the ability of culture supernatants from V. anguillarum and Salmonella enterica serovar Typhimurium (Table 1) siderophore-defective mutants to cross-feed different indicator strains that were defective in DHBA and/or siderophore synthesis. It is known that vanchrobactin can substitute the function of enterobactin to supply iron to enterobactin-deficient mutants (Lemos et al., 1988; Conchas et al., 1991). Two mutants of S. enterica serovar Typhimurium LT2 (Pollack et al., 1970), which are deficient in two different biosynthetic steps of enterobactin production, were used to detect vanchrobactin and DHBA production: (i) strain enb-7, which can use exogenous enterobactin, vanchrobactin or DHBA to overcome iron-limiting conditions; and (ii) enb-1, which can use only enterobactin or vanchrobactin for iron supply (Conchas et al., 1991; Lemos et al., 1988).
We designed two cross-feeding assays: (i) V. anguillarum MB10 vabC-defective mutant was used as an indicator strain to test MB3(vabE), MB14(vabF), and S. enterica enb-7 and enb-1; (ii) enb-7 and enb-1 were used as indicators to test MB10(vabC), MB3(vabE) and MB14(vabF) mutants. In each assay, strain RV22 was used as positive control. Each indicator strain was inoculated into CM9 minimal medium containing a concentration of 2,2'-dipyridyl that was higher than the MIC for that strain. Strains to be tested were cultured on LB agar, and cells were harvested with a sterile loop, and placed on the surface of the indicator-strain plates. The results were scored as positive when tester cells promoted the growth of indicator strains.
Phylogenetic tree construction.
A total of 410 adenylation-domain (A-domain) sequences were retrieved from UniProt/TrEMBL/SWISS-PROT databases (Apweiler et al., 2004; Boeckmann et al., 2003). These sequences were aligned with the A-domain sequences predicted for V. anguillarum RV22 VabE (A-domain) and VabF (A1- and A2-domains) proteins, using CLUSTAL W software (Thompson et al., 1994). The resulting alignments were visually inspected, and, when appropriate, manually adjusted. The phylogenetic tree was built using the amino acid sequence that contained the A-domain substrate-binding pocket (positions 190–331) (Stachelhaus et al., 1999). Phylogenetic trees relied on the modules SEQBOOT, PROTDIST, NEIGHBOR and CONSENSE of the PHYLIP package (Felsenstein, 1996). Bootstrap analysis was used to assess statistical support for relationships via branch and bound analysis of 1000 replicated datasets. The tree obtained with the initial 413-sequence multiple alignment was further used to select 56 aa sequences that grouped together with our working sequences. These were employed to construct a second phylogenetic tree, with the same methodology described above.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Lemos et al., 1988), in contrast with the well-known pJM1-mediated system described for V. anguillarum serotype O1 strain 775 (Crosa & Walsh, 2002; Stork et al., 2002). Sequence analysis of an 18 kb chromosomal fragment from V. anguillarum strain RV22 (see Methods) revealed the presence of eight ORFs, named dahP, vabA, vabC, vabE, vabB, vabS, vabF and vabH (Fig. 1), whose deduced protein products showed significant similarity to proteins involved in siderophore-mediated iron utilization in other bacterial species (Table 3). While DahP and VabB showed 69 and 97 % identity, respectively, to partially sequenced DahP and AngBch serotype O1 chromosomally encoded homologues (Alice et al., 2005), the VabA, VabC, VabE proteins showed an identity higher than 97 % to AngAch, AngCch and AngEch, respectively, which are encoded in the chromosome of V. anguillarum serotype O1 775 strain, and are involved in the synthesis of DHBA (Alice et al., 2005) (Table 3). The same gene arrangement was observed for the RV22 and 775 homologues, and a putative Fur box predicted within the vabA–vabC intergenic region was found to be 100 % identical to that reported between angAch and angCch (Fig. 1). The three remaining genes of this cluster, vabS, vabF and vabH, are transcribed in the same DNA strand as vabB (with a putative Fur box identified upstream of vabH); homologues of these genes have not been described to date in V. anguillarum (Table 3), and they are analysed in detail below.
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vabA), MB11(vabB) and MB10(vabC) mutants were severely affected in their ability to grow, in comparison with the parental strain (Fig. 2a).
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). In addition, analysis of the supernatants by the Arnow test revealed that DHBA production was completely abolished in MB12 and MB11 mutants, and reduced about sixfold in the MB10 mutant (Fig. 2b). The cross-feeding assays showed that V. anguillarum RV22 wild-type and S. enterica enb-1, but not enb-7, promoted the growth of the vabC mutant (Table 4). Similarly, supernatants of the vabC mutant failed to complement enb-7. Together, these results indicate that vabA, vabB and vabC are most probably involved in the synthesis of DHBA in V. anguillarum RV22, and that they appear to be necessary for siderophore production, and growth under iron-limited conditions.
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). An analogous situation was reported for V. anguillarum serotype O1, when an angC mutant was found to produce residual amounts of DHBA, but this production was abolished in an angC–menF double mutant (Alice et al., 2005).
When MB10 and MB12 mutants were complemented with plasmids pMB13 (containing the vabC gene) and pMB14 (containing the vabA gene), respectively, the growth levels under iron-restricted conditions were restored to the wild-type level (Fig. 2a), indicating that vabC and vabA are essential for iron uptake. In addition, the complemented mutants showed levels of DHBA and siderophore production comparable with those observed in the parental strain (Fig. 2b), confirming that vabC and vabA are essential for DHBA and siderophore biosynthesis in strain RV22.
Characterization of vabE and vabF, and prediction of vanchrobactin components
In catechol-type siderophores, DHBA must be activated by a 2,3-dihydroxybenzoate-AMP ligase. The remaining siderophore components are further selected and activated by non-ribosomal peptide synthetases (NRPSs), and assembled with DHBA to form the final siderophore molecule (Earhart, 1996; Walsh et al., 1990). The RV22 VabE sequence is nearly identical to AngEch, which is encoded by the chromosome of the V. anguillarum O1 strain 775. AngEch is a 2,3-dihydroxybenzoate-AMP ligase involved in the synthesis of the siderophore anguibactin, and it is responsible for the selection and activation of DHBA (Alice et al., 2005). On the other hand, VabF (with a predicted size of 2835 residues) is homologous to members of the NRPS family (Table 3) that are involved in the final assembly of siderophores, for instance E. coli EntF (Rusnak et al., 1991), Chromobacterium violaceum CbsF, and Acinetobacter baumannii DhbF-like proteins (Dorsey et al., 2003) (Table 3).
To ascertain the role of VabE and VabF in vanchrobactin synthesis in RV22, we constructed in-frame deletion mutants of vabE and vabF. As shown in Fig. 2a, both mutants were significantly impaired for growth under iron-limited conditions, and siderophore levels in supernatants were less than 20 % of those detected in the parental strain, indicating that these mutations result in a severe reduction of siderophore synthesis (Fig. 2b). These results are comparable with those obtained for vabA, vabB and vabC mutants. However, analysis of the supernatants using the Arnow test showed a significant production of DHBA (about 50 % of that produced by the wild-type) in vabE- and vabF-null mutants (Fig. 2b). DHBA production in vabE and vabF mutants was verified in cross-feeding bioassays using the enterobactin-deficient S. enterica enb-7 as the indicator strain. Results showed that both vabE and vabF mutants could promote the growth of enb-7, but not enb-1, indicating that vabE and vabF mutants produce DHBA, but are unable to synthesize vanchrobactin (Table 4). Complementation of vabF mutation with the parental vabF gene, provided in trans (plasmid pMB15), restored the wild-type phenotype in growth ability and siderophore production (Fig. 2a, b).
These results are in agreement with the hypothesis that VabE and VabF are two NRPSs involved in the final steps of vanchrobactin assembly. Since these steps are subsequent to the production of DHBA, this molecule would be produced normally in vabE and vabF mutants, but it could not be used for the synthesis of vanchrobactin.
Since VabE and VabF are homologous to well-described NRPSs involved in siderophore biosynthesis, and these enzymes exhibit a modular organization (Crosa & Walsh, 2002), we subjected the amino acid sequences of VabE and VabF to a domain-prediction analysis. Results indicated that VabE contains a unique A-domain, and that VabB consists of a single aryl-carrier-protein (ArCP) domain. Therefore, VabE and VabB could constitute an initiation module with the A–ArCP structure. VabF shows a seven-domain structure, organized into two modules: the N-terminal module (VabF module 1) follows the C–A–PCP (condensation–adenylation–peptidyl-carrier-protein) domain structure, whereas the C-terminal module (VabF module 2) contains a C–A–PCP–TE (condensation–adenylation–peptidyl-carrier-protein–thioesterase) domain structure. In addition, we were able to locate the conserved core motifs of the catalytic domains (Marahiel et al., 1997) in each of the VabE, VabB and VabF predicted domains (data not shown).
In order to elucidate VabE and VabF substrate specificity, we applied the non-ribosomal code (Challis et al., 2000; Stachelhaus et al., 1999). A sequence alignment of the predicted adenylation domains of VabE and VabF with the well-characterized adenylation domain of the Bacillus subtilis gramicidin synthetase A (GrsA) was carried out to locate the 10 residue positions that are crucial for substrate binding. Results of this analysis suggest that either DHBA or salicylate, and serine, are activated by VabE and VabF, respectively. However, it seems clear that DHBA should be the actual substrate activated by the initiation module (VabE–VabB), not only because the closest VabE homologues are 2,3-dihydroxybenzoate-AMP ligases (Table 3), but also because vanchrobactin is a catechol-type siderophore (Lemos et al., 1988; Soengas et al., 2006).
Since the sequence alignments were not sufficient to establish VabF substrate-specificity prediction, we constructed a neighbour-joining tree from a multiple sequence alignment of A-domain substrate-binding pocket sequences for substrate determination (Di Vincenzo et al., 2005). The results confirmed our previous specificity predictions for VabE and VabF. The VabE A1-domain was clustered with a bootstrap value of 66 % into a branch containing DHBA and salicylate-specific A-domains (Fig. 3). This cluster could be further divided in two subgroups: one including some well-characterized proteins with DHBA-AMP ligase activity, such as E. coli EntE or Vibrio cholerae VibE; and a second subgroup including proteins with salicyl-AMP ligase activity. The VabE A-domain was clustered with a bootstrap value of 68 % into the subgroup of 2,3-dihydroxybenzoate-AMP ligases. Interestingly, the VabF A1-domain clustered together with a bootstrap value of 95 % into the branch that grouped A-domains responsible for the selection and adenylation of amino acids with a positively charged side chain. The VabF A2-domain clustered, with a bootstrap value of 96 %, with serine-specific A-domains, such as those of the E. coli and Shigella sonnei EntF proteins, and the A-1 and A-2 domains of Pseudomonas syringae SyrE (Fig. 3).
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). The recent elucidation of vanchrobactin chemical structure (Soengas et al., 2006) showed that this siderophore is a N-[N'-(2,3-dihydroxybenzoyl)-arginyl]-serine (Fig. 4c). Thus, the structural chemical data are in complete agreement with the component prediction based on the amino acid sequences of VabE and VabF. These data, together with the mutagenesis analysis described above, strongly suggest that VabE and VabF are the key enzymes involved in vanchrobactin biosynthesis.
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), such as the E. coli EntS (P43), a protein encoded within the ent operon that participates in the secretion of enterobactin (Furrer et al., 2002). The VabS protein shares conserved domains with members of the MFS family, especially the amino acid motifs that define it (Pao et al., 1998).
The prediction of transmembrane structure of V. anguillarum VabS using the TMHMM program showed the characteristic 12-transmembrane segments, equivalent to those described for the E. coli enterobactin siderophore exporter P43 (EntS) (Furrer et al., 2002), with the exception of transmembrane domain 11. An analogous situation was reported for the A. baumannii P45 protein (Dorsey et al., 2003).
In order to gain an insight into the role of vabS in vanchrobactin-mediated iron acquisition, we constructed a deletion mutant. The vabS mutant showed a reduction in its growth ability under iron-limited conditions (Fig. 2a). Interestingly, when the Arnow assay was carried out with vabS mutant supernatants, not only did it yield values similar to the RV22 wild-type during the first hours of culture (data not shown), but these values showed an increase of 50 % after 22 h, with respect to the wild-type (Fig. 2b). However, the levels of siderophore production in the vabS mutant, as determined by the CAS assay, did not show significant differences with respect to the wild-type (Fig. 2b). In this regard, it has been reported that the E. coli P43 mutant is unable to secrete enterobactin efficiently, its growth ability being supported by 2,3-dihydroxybenzoyl-serine (DBS), an enterobactin breakdown product that retains iron-chelating activity, and freely diffuses out of the cells (Furrer et al., 2002; Hantke, 1990; Rabsch et al., 2003). Something similar could occur in V. anguillarum RV22, and a precursor or breakdown product of vanchrobactin could also act as a siderophore. These products could be detected by Arnow and CAS assays, although their efficiency in supplying iron to the cell would be inferior to the natural siderophore, and would probably result in lower growth levels (Fig. 2a). If the cells have not taken up enough iron, this would produce an increase in DHBA and/or other vanchrobactin precursors (Fig. 2b). These results, together with the comparative analysis of the protein sequence, suggest that VabS could be involved in the export of vanchrobactin.
vabH encodes a putative siderophore-degrading enzyme
In most bacteria, once a ferric–siderophore complex enters the cytoplasm, iron is removed by a mechanism requiring an esterase activity. To test the potential role of VabH in siderophore utilization, we constructed an in-frame deletion mutant of the vabH gene. As shown in Fig. 2a, the MB19(vabH) null mutant was impaired for growth under iron-limited conditions, although both the CAS assay and the Arnow test showed a significant production of siderophore and DHBA, respectively (Fig. 2b). This could be evidence of the inability of the vabH mutant to utilize intracellular ferric–vanchrobactin. When the MB19 mutant strain was transformed with the pMB16 plasmid containing the vabH gene, the growth ability under iron-limited conditions was restored to wild-type levels. In addition, DHBA production and siderophore production in the complemented mutant gave values that were similar to those obtained with the parental strain (Fig. 2a).
In E. coli, the internalized ferric–enterobactin complex is degraded to ferrous iron (Fe2+) and the linear trimer, dimer and monomer of DBS by means of a ferric–enterobactin esterase (Fes) (Brickman & McIntosh, 1992; Furrer et al., 2002). The predicted translation product of vabH is a protein of 401 aa, with 53 % similarity to Fes (Table 3), suggesting that this gene could be the E. coli fes orthologue. However, other Fes homologues do not play a role in iron release from the ferric–siderophore, but they do play a role in the degradation of the siderophore. This is the case for CbsH, a Fes homologue with a peptidase activity involved in degradation of chrysobactin, which is a siderophore produced by Erwinia chrysanthemi that has a structure closely similar to vanchrobactin (Rauscher et al., 2002). Thus, although more work is needed to unravel the precise role of this gene in vanchrobactin utilization, V. anguillarum VabH could play a potential role in degrading vanchrobactin after its internalization.
A biosynthetic model for vanchrobactin
From the genetic data reported here, as well as the chemical structure and previous reports for enterobactin and anguibactin synthesis (Crosa & Walsh, 2002), we propose the model for the biosynthesis of vanchrobactin shown in Fig. 4.
DAHP synthase is not one of the enzymes directly connected to siderophore synthesis, but catechol-type siderophore biosynthesis itself starts with the conversion of chorismate into the DHBA, and DAHP is an intermediate in chorismate synthesis (Kloosterman et al., 2003). According to the role assigned to the E. coli homologues (Earhart, 1996; Walsh et al., 1990), the enzymes involved in DHBA biosynthesis from chorismate in V. anguillarum RV22 should be an isochorismate synthase (VabC), an isochorismatase (VabB), and a 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (VabA) (Fig. 4a).
Once DHBA is synthesized, 2,3-dihydroxybenzoate-AMP ligase activity is necessary for DHBA activation (Crosa & Walsh, 2002). The protein encoded by vabE is the candidate for activating DHBA to its acyl-adenylate derivative in an ATP-dependent manner, and for transferring this activated species to holo-VabB. According to enterobactin and anguibactin synthesis models (Ehmann et al., 2000), VabB could be involved in DHBA biosynthesis through its isochorismatase activity, and in further binding of DHBA through its ACP domain.
The adenylation domains of NRPS are responsible for the selection and incorporation of either one amino acid or one carboxy acid into the final product (Crosa & Walsh, 2002). According to the sequence analysis and chemical data mentioned above, the VabF A1-domain should activate an arginine residue, and, through its PCP domain, the 2,3-dihydroxybenzoyl-arginine precursor should be synthesized. The VabF A2-domain should then activate a serine residue, since this is the substrate predicted by the non-ribosomal code, and on the basis of the high homology showed by the VabF A2-domain and E. coli EntF (Fig. 3), which is a well-characterized ATP-dependent serine-activating enzyme (Reichert et al., 1992). The TE domain is the last domain to play a role in the biosynthesis of a non-ribosomal peptide, releasing the final product (Lautru & Challis, 2004). The TE domain predicted in the VabF module 2 would have this role, releasing vanchrobactin into solution.
In conclusion, from the results described here and those reported by others (Alice et al., 2005), it seems reasonable to speculate that the vanchrobactin biosynthesis cluster, or at least part of it, is shared by serotypes O1 and O2 of V. anguillarum, and that vanchrobactin is probably the functional siderophore in strains lacking the pJM1-type plasmid. A study of the distribution of the genes described here is required for other strains in order to clarify the evolution of plasmid- and chromosomal-mediated siderophore biosynthesis in V. anguillarum.
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ACKNOWLEDGEMENTS |
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Received 11 July 2006;
revised 16 August 2006;
accepted 17 August 2006.
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