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Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela 15782, Galicia, Spain
Correspondence
Manuel L. Lemos
mlemos{at}usc.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequences reported in this paper is AJ699306.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The ability to obtain iron from free haem or haem proteins from the host tissues constitutes an important source of iron for many bacterial pathogens (Genco & Dixon, 2001; Osorio & Lemos, 2002). However, one of the main strategies to obtain iron is the synthesis and secretion of Fe(III) chelators, named siderophores, which are low molecular mass iron-chelating molecules that can remove iron from host iron-binding proteins (Crosa, 1989) and then enter the cell through outer-membrane receptor proteins. Many siderophores are small peptides synthesized by non-ribosomal peptide synthetases (NRPSs), which are multimodular enzymes that produce peptide products of a particular sequence without an RNA template (Crosa & Walsh, 2002; Schwarzer et al., 2003).
Iron acquisition has been widely recognized as a determinant of bacterial virulence (Braun, 2005). Thus, it is not surprising that several iron-transport systems have been described as part of horizontally transmitted elements, showing differential occurrence in pathogenic vs non-pathogenic strains. The siderophore aerobactin genes are found on plasmids in certain strains of Escherichia coli and Salmonella (Williams, 1979; Colonna et al., 1985), but have been described to constitute a chromosomal pathogenicity island in Shigella flexneri strains (Vokes et al., 1999). Similarly, highly pathogenic strains of Yersinia (Yersinia enterocolitica 1B, Yersinia pseudotuberculosis I and Yersinia pestis) harbour a large chromosomal fragment termed the high-pathogenicity island (HPI), which is absent from low-pathogenic and avirulent strains (Carniel et al., 1996). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin and is integrated in the chromosomal asn-tRNA gene (Buchrieser et al., 1998). It consists of a 30.5 kb highly conserved functional core region and a 5–13 kb AT-rich variable part (Rakin et al., 1999). The core region comprises genes for the biosynthesis (irp1–irp5 and irp9) and uptake (irp6–irp8 and fyuA) of the siderophore, as well as an AraC-like regulator (ybtA). An integrase gene (intB) is found at the upstream end of the HPI. Specific studies conducted with mutant strains have demonstrated the importance of the yersiniabactin system in the virulence of Y. enterocolitica (Rakin et al., 1994), Y. pestis (Bearden et al., 1997) and Y. pseudotuberculosis (Carniel et al., 1992). Although first identified in Yersinia spp., the HPI has been also described in several genera of Enterobacteriaceae (Schubert et al., 1998; Bach et al., 2000; Olsson et al., 2003).
Photobacterium damselae subsp. piscicida was initially isolated from a massive fish mortality in Chesapeake Bay, and the disease was named pasteurellosis after the classification of this agent as Pasteurella piscicida (Janssen & Surgalla, 1968). The pathogen was later reassigned to the genus Photobacterium as Photobacterium damselae subsp. piscicida, sharing species epithet with P. damselae subsp. damselae (formerly Vibrio damsela) (Gauthier et al., 1995) and thus constituting a new member of the family Vibrionaceae. P. damselae subsp. piscicida is one of the most devastating bacterial pathogens in marine aquaculture, due to its wide geographical distribution, host range and massive mortality (Magariños et al., 1996a). The virulence of this micro-organism is believed to be a multifactorial process not yet fully understood. The major virulence factor so far identified is a plasmid-encoded protein, AIP56, which induces apoptosis in fish macrophages and neutrophils (do Vale et al., 2005). It is also believed that polysaccharide capsular material constitutes a main virulence factor (Magariños et al., 1996b). This bacterium is able to obtain iron from haemin and haemoglobin as unique iron sources in vitro (Magariños et al., 1994), and a genetic system for haem uptake has been recently described (Juiz-Río et al., 2005a). A gene encoding the ferric uptake regulator (Fur) protein has been identified in this species, and it is believed to act as an iron-dependent transcriptional repressor regulating genes involved in iron uptake (Juiz-Río et al., 2004; Osorio et al., 2004). It was reported that strains of this subspecies can efficiently obtain iron from human transferrin, and this is achieved by a mechanism involving the production of siderophores (Magariños et al., 1994). However, the nature of this siderophore is still unknown and the genes involved in its biosynthesis have not been characterized, although preliminary chemical assays and bioassays indicated that it is neither a catechol nor a hydroxamate (Magariños et al., 1994).
This study was undertaken to obtain an insight into the genetic basis of the siderophore-mediated iron-uptake system of P. damselae subsp. piscicida, to study its role in virulence for fish and to assess if genetic differences exist among P. damselae subspecies and strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. damselae subsp. piscicida and P. damselae subsp. damselae strains were routinely grown at 25 °C in either Tryptic Soy Agar or Tryptic Soy Broth (Difco) supplemented with 1 % NaCl (TSA-1 and TSB-1, respectively), and in CM9 minimal medium (Mouriño et al., 2004). E. coli strains were grown at 37 °C in Luria–Bertani (LB) broth or LB agar, as well as in CM9 minimal medium, supplemented with antibiotics when appropriate. Y. enterocolitica strains were grown at 37 °C in LB, Nutrient Broth (NB) and Nutrient Agar (NBA). All strains were stored frozen at –80 °C in LB broth with 20 % glycerol. Antibiotics were used at the following final concentrations: kanamycin (Kan) at 25 µg ml–1; ampicillin sodium salt (Amp) at 50 µg ml–1; chloramphenicol (Cam) at 20 µg ml–1; rifampicin (Rif) at 50 µg ml–1. All stocks were filter-sterilized and stored at –20 °C. The iron chelator 2,2'-dipyridyl (Sigma) was prepared at 10 mM in water.
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). In brief, tester (DI21) and driver strain (RM71) genomic DNA (2 µg) were each digested with 10 units RsaI for 5 h. The tester DNA was aliquoted into two tubes, and each was ligated to a different adaptor provided with the kit. Two hybridization rounds were carried out: in the first hybridization, an excess of driver DNA was added to each adaptor-ligated tester DNA, and samples were then heat denatured and allowed to anneal. In the second hybridization round, the two samples from the first hybridization were mixed together without denaturation. The product of this last hybridization was then used as a template in a PCR reaction to amplify the tester-specific sequences, using Advantage cDNA polymerase mix (Clontech). The obtained PCR products were cloned into the pGEM-T Easy vector (Promega) and recombinant clones were screened by restriction analysis prior to DNA sequencing.
Recombinant DNA techniques, DNA sequencing and data analysis.
Recombinant DNA methods were performed following standard protocols (Sambrook & Russell, 2001). Chromosomal DNA was isolated using the Easy-DNA kit (Invitrogen). Plasmid purification and elution of DNA fragments from agarose gels were performed using kits from Qiagen. Southern blot analysis was performed with Hybond-N+ membranes (Amersham Biosciences), using the ECL Direct Nucleic Acid labelling and detection system (Amersham Biosciences) and following the manufacturer's instructions. PCR reactions were carried out using the Expand Long Template kit (Roche). For inverse PCR, chromosomal DNA was digested with a single restriction enzyme, the fragments self-ligated and the ligation products subsequently used as templates in PCR reactions with suitable primers. DNA sequences were determined by the dideoxy chain-termination method using the CEQ DTCS-Quick Start Kit (Beckman Coulter) using a capillary DNA sequencer CEQ 8000 (Beckman Coulter). Comparison of the sequence data with published sequences in EMBL/GenBank was performed with the BLAST software via the internet (http://www.ncbi.nlm.nih.gov/blast and http://www.ebi.ac.uk/blast/index.html). Prediction of protein domains was carried out by using the Pfam database online facilities (http://www.sanger.ac.uk/Software/Pfam/). Putative transcriptional terminators were predicted using RNAMotif (Macke et al., 2001).
RNA isolation and RT-PCR.
P. damselae subsp. piscicida DI21 was grown in TSB-1 and then subcultured on CM9 minimal medium. Total RNA was prepared from mid-exponential-phase cultures using the RNAwiz Isolation Reagent (Ambion), according to the manufacturer's instructions. RNA preparations were then subjected to standard treatments with RNase-free DNase I. Reverse transcription reaction was performed by using the M-MLV reverse transcriptase (Invitrogen). For each reaction, 1 µg total RNA was used. Subsequent PCR amplification was carried out with Taq polymerase (Bioline) using suitable primers. As negative controls, DNA contamination of the RNA samples was ruled out by PCR using Taq DNA polymerase without reverse transcriptase. As positive controls, each primer combination was tested in PCR reactions carried out using chromosomal DNA as template.
Protein analysis.
P. damselae subsp. piscicida DI21 and CS31 and P. damselae subsp. damselae RM71 cells were grown in CM9 supplemented with either FeCl3 10 µM or 2,2'-dipyridyl 60 µM (iron-sufficient and iron-restricted conditions, respectively). This concentration of 2,2'-dipyridyl was lowered to 30 µM in the case of the DI21 irp1 mutant strain (CS31). Cells were centrifuged and total membrane proteins were obtained as previously described (Toranzo et al., 1983). Protein concentration was adjusted for all the samples, and proteins were separated by SDS-PAGE. The protein bands were visualized by staining with Coomassie brilliant blue.
Construction of an irp1-targeted disruptant of P. damselae subsp. piscicida.
An EcoRI–XbaI fragment of 4672 bp, which included 
1500 bp of the irp2 downstream end and 3100 bp of the irp1 upstream end (Fig. 1a), was ligated into vector pWKS30. The DNA insert was further excised with NotI–SalI and ligated into the suicide vector pNidKan (Mouriño et al., 2004), to generate pSJR52. As a pCVD442 derivative, pNidKan contains R6K ori, requiring the pir gene product for replication. Insertion of the suicide vector into the chromosome by a single crossover results in a KanR phenotype. The pSJR52 plasmid was transformed into E. coli S17-1 
pir followed by mobilization into DI21-Rif. Insertional mutants were selected on agar plates containing kanamycin (50 µg ml–1) and rifampicin (50 µg ml–1), and presence of the inserted plasmid into the irp1 gene was confirmed by Southern hybridization (data not shown).
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in solid medium. The assay was performed by spotting 20 µl supernatant of each bacterial culture, previously grown in CM9, onto chrome azurol S (CAS) agar plates. The ability of P. damselae subsp. piscicida strains to grow under conditions of iron limitation was evaluated by using CM9 minimal medium, in the presence of 60 µM 2,2'-dipyridyl (Sigma).
The pathogenicity of P. damselae subsp. piscicida DI21-Rif and CS31 strains for turbot (Scophthalmus maximus) was assayed using 10 fish (mean weight 15 g) per dose, as previously described (Magariños et al., 1992). Fish were inoculated with bacterial doses ranging from 103 to 107 cells. Mortalities were recorded daily for 10 days, and the LD50 was calculated according to Magariños et al. (1992).
Cross-feeding assays.
Cross-feeding assays were utilized to test the ability of Y. enterocolitica and P. damselae subsp. piscicida strains to induce the growth of the same strains subjected to iron starvation. Two mutants of Y. enterocolitica that were deficient in the biosynthesis of yersiniabactin (strain WA-CS irp1 : : Kanr), or in the yersiniabactin receptor (strain WA fyuA2), respectively, were used. P. damselae subsp. piscicida CS31 was used for detection of yersiniabactin utilization. One hundred microlitres of overnight cultures of each indicator strain was added to 3 ml molten TSA-1 (P. damselae subsp. piscicida strains) or NBA (Y. enterocolitica strains) and plated onto appropriate prepoured TSA or NBA plates supplemented with 200 µM 2,2'-dipyridyl. Sterile filter-paper discs were loaded with 10 µl of cultures of different strains previously grown in TSC-1 (P. damselae subsp. piscicida) or NB (Y. enterocolitica) supplemented with 60 and 100 µM 2,2'-dipyridyl, respectively, to induce siderophore production. Discs loaded with 10 µl yersiniabactin (0.1 mg ml–1) were also tested. Results were scored as positive or negative after 48 h incubation. Yersiniabactin was purchased from EMC microcollections GmbH.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, siderophore assays specific for catechols or hydroxamates did not detect the siderophore produced by P. damselae subsp. piscicida DI21 (Magariños et al., 1994). We hypothesized that the production of different types of siderophore would be due to a differential gene content between these two strains. Therefore, we carried out SSH in order to isolate DNA fragments encountered uniquely in DI21 (tester strain), but absent from RM71 (driver strain). Some of the subtracted clones contained DNA sequences encoding transposases, tRNA hydroxylases and hypothetical proteins (to be published elsewhere). One clone, pSSH1, contained a 1500 bp insert that encoded a 508 amino acid partial protein with significant similarity (63 %) to the Y. enterocolitica high molecular mass protein 2 (HMWP2) (Table 2), a member of the family of the NRPSs involved in the synthesis of siderophore yersiniabactin. Based on this homology, this P. damselae subsp. piscicida gene was named irp2. Southern blot hybridization demonstrated that the DNA fragment cloned in pSSH1 was absent from the driver strain, RM71 (data not shown).
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9 kb XbaI–XbaI DNA fragment (data not shown). This DNA fragment was further cloned into pGEM-T Easy and sequenced to completion and the complete sequence of irp2 was established as an ORF encoding a protein of 1979 aa with a predicted molecular mass of 221 kDa. Inverse PCR was used to amplify additional DNA segments upstream and downstream of irp2, which were cloned and sequenced to completion. A gene cluster (irp cluster) spanning 35 kb and including 11 ORFs was thus identified (Fig. 1a
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Predicted protein sequences
Homologues of the proteins encoded by the irp cluster predicted ORFs are summarized in Table 2. The first ORF, dahP, encodes a putative 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. It showed the highest similarity with a DAHP synthase of Vibrio vulnificus. Homologues of this enzyme are responsible for the condensation of the pentose phosphate pathway intermediate D-erythrose 4-phosphate and the glycolytic pathway intermediate phosphoenolpyruvate to DAHP, a step in the biosynthesis of chorismate. The second and third genes of this cluster were termed araC1 and araC2, respectively. The closest relatives of AraC1 and AraC2 proteins were putative AraC-family transcriptional regulators of Photobacterium profundum and Vibrio cholerae RC385. Homologues also included putative transcriptional regulators of other bacterial species, but the homology was restricted to the C-terminus. A protein domain prediction by using the Pfam database identified a helix–turn–helix motif within approximately the last 100 residues of the C-terminus of the deduced AraC1 and AraC2 proteins (data not shown), which is a conserved domain typical of this family of regulators. Interestingly, AraC2 showed similarity to YbtA, a transcriptional activator that controls expression of genes within the Yersinia HPI (Fetherston et al., 1996; Anisimov et al., 2005).
The fourth ORF of the cluster was termed frpA, encoding a predicted 660 amino acid protein that showed significant similarity with the Y. pestis and Pseudomonas fluorescens yersiniabactin and quinolobactin outer-membrane receptors, respectively. The fifth ORF, irp8, encodes a protein that showed homology to putative proteins whose function has not been ascertained in any species. A search conducted using the COG database (http://www.ncbi.nlm.nih.gov/COG) placed this protein within the group of permeases of the major facilitator superfamily (MFS). Similarity was found between this protein and YbtX, a highly hydrophobic cytoplasmic membrane protein encoded in the Yersinia HPI (Fetherston et al., 1999). Its subcellular location and the similarity to MFS proteins suggest that Irp8 could be involved in siderophore export.
The sixth ORF of the cluster corresponds to the irp2 gene, isolated by subtractive hybridization between strains DI21 and RM71 (see above). The seventh ORF was termed irp1; its start codon was located 50 bp downstream of the stop codon of irp2. Irp1 is a protein of 3996 aa, with a predicted molecular mass of 442 kDa. The first N-terminal 430 aa of Irp1 show homology to typical PKS (polyketide synthase) domains, and the identity percentage in this region was 62 % to Y. enterocolitica HMWP1.
The eighth ORF was termed irp3; it encodes a protein with homology to thiazolinyl reductase components of the siderophore yersiniabactin biosynthesis pathway (YbtU component) (Miller et al., 2002). The protein encoded by the ninth ORF, Irp4, was found to be 43 % and 41 % identical to Y. pestis YbtT and Y. enterocolitica Irp4, respectively, described as thioesterase-like proteins involved in yersiniabactin biosynthesis (Geoffroy et al., 2000; Pelludat et al., 1998), and 36 % identical to a thioesterase-like protein located in the anguibactin biosynthetic gene cluster of Vibrio anguillarum (Farrell et al., 1990).
The tenth ORF encodes a protein which we termed Irp9. It is similar to Y. enterocolitica Irp9, an enzyme that directly converts chorismate into salicylate, which is further used as a precursor in the synthesis of yersiniabactin (Pelludat et al., 2003). Irp5, encoded by the eleventh ORF, was found to be homologous to a series of aryl-activating enzymes, involved in the adenylation and activation of aryl groups and amino acids. It is 47 % identical to Y. pestis YbtE, a salicyl-AMP ligase which activates salicylic acid and transfers the activated molecule to the first ArCP (aryl-carrier protein) domain of the non-ribosomal peptide synthetase HMWP2 (Gehring et al., 1998). Interestingly, Irp5 is 44 % identical to V. cholerae VibE, the vibriobactin-specific 2,3-dihydroxybenzoate-AMP ligase, the enzyme that adenylates and activates 2,3-dihydroxybenzoate, a precursor of vibriobactin (Wyckoff et al., 1997).
Transcriptional organization of the cluster
The 11 genes of the cluster described in this study have the same transcriptional polarity. Downstream of irp5 is an ORF with opposite transcriptional polarity to the 11-gene cluster described and its predicted protein product shows similarity to putative inner-membrane ATPases (data not shown). Upstream of dahP we sequenced 3 additional kb, but no ORFs were found.
Sequence analysis identified a conserved putative Fur-binding site (Fur box), upstream of dahP gene, which shares 17 out of 19 identical nucleotides with the E. coli Fur-box consensus sequence. Similarly, a potential Fur box with identity in 16 of 19 positions with the consensus was found upstream of araC2 (Fig. 1a). This would suggest the existence of at least two iron-regulated promoters within this cluster. To study the transcriptional organization of the cluster, we conducted a series of reverse-transcriptase reactions with primers targeted to the downstream ends of irp5 (RT-1), araC2 (RT-2) and araC1 (RT-3), and subsequent PCR reactions were performed with three different primer combinations, A, B and C, that amplified internal fragments of irp1 (653 bp), araC2 (407 bp) and dahP (470 bp), respectively (Fig. 1a). All the reactions yielded a product of the expected size, and negative and positive controls (see Methods) corroborated the accuracy of the RT-PCR results (Fig. 1b). These results demonstrate that the 11 genes of the cluster described in this study can be co-transcribed from the promoter upstream of dahP, although the existence of additional promoters, particularly upstream of araC2, cannot be ruled out. When we applied the RNAMotif program, a unique transcriptional stop was predicted, starting at 18 bp downstream of the irp5 stop codon (Fig. 1a). No additional transcriptional terminators were predicted within this gene cluster.
Presence of iron-regulated high molecular mass proteins in P. damselae subsp. piscicida DI21
The presence of Fur-binding sequences similar to the E. coli consensus upstream of dahP and araC2 would suggest that iron concentration plays a role in regulating the expression of genes within the irp cluster. P. damselae subsp. piscicida DI21 cells were grown in iron-rich and iron-limited media, and the profiles of total membrane proteins were compared. As a result, two high molecular mass protein bands were observed when the cells were cultured in iron-restricted media, whereas these bands were not observed under conditions of iron sufficiency (Fig. 2). The molecular masses of these two proteins likely correspond to those predicted for the Irp1 (442 kDa) and Irp2 (221 kDa) as described above, encoded by the irp cluster. These two protein bands were absent under both iron-limiting and iron-sufficient conditions in the P. damselae subsp. damselae strain RM71, which was used as the driver strain in the SSH experiment described above. Although the subcellular location of Irp1 and Irp2 homologues is not well known, there is some evidence that they may contain hydrophobic domains that may be membrane associated (Guilvout et al., 1993) and this could explain the purification of these proteins in the membrane fraction.
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), which are modularly organized multi-enzyme complexes that represent at the same time template and biosynthetic machinery (Schwarzer et al., 2003). Using the Pfam database, we found that the predicted domain organizations of the P. damselae subsp. piscicida Irp5, Irp2 and Irp1 proteins are highly similar to those predicted for the Y. enterocolitica Irp5, HMWP2 and HMWP1 proteins. However, subsp. piscicida Irp2 lacks an ArCP domain, which is otherwise encountered at the N-terminus of Y. enterocolitica HMWP2. Interestingly, Irp1 is larger than HMWP1 and contains different domains (Fig. 3).
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; Schwarzer et al., 2003). Thus, it is possible to predict the substrates that will be recognized and adenylated by the three A domains predicted in this study: one in Irp5, and one in each of Irp2 and Irp1. We carried out a sequence alignment of these three predicted A domains with that of the gramicidin S synthetase (GrsA) from Bacillus brevis and determined the ten conserved residues of the substrate-binding pocket. As shown in Table 3, the Irp5 A domain is predicted to select salicylate, thus having the same substrate specificity as Y. enterocolitica Irp5. On the other hand, the A domains of Irp2 and Irp1 are predicted to select and activate cysteine, which is the same substrate as the homologous A domain present in Y. enterocolitica HMWP2.
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Parental and mutant strains showed a similar growth in CM9 minimal medium supplemented with 10 µM FeSO4 (iron-sufficient conditions) (Fig. 4). The same strains were grown in CM9 in the presence of the iron chelator 2,2'-dipyridyl at a concentration of 60 µM. Under these iron-limiting conditions, the irp insertional mutant CS31 was impaired for growth with respect to the parental strain (Fig. 4). In addition, when the protein profiles of CS31 and the parental strain, under both iron-sufficient and iron-limiting conditions, were compared, the mutant lacked the high molecular mass iron-regulated protein believed to correspond to Irp1 (Fig. 2).
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100-fold higher than with the parental strain (8x105), demonstrating that production of siderophores mediated by the irp cluster contributes significantly to the virulence of P. damselae subsp. piscicida for fish.
Cross-feeding assays
Based on protein-domain sequence predictions, the siderophore synthesized by P. damselae subsp. piscicida might be similar to yersiniabactin. To assess this possibility, P. damselae subsp. piscicida DI21 and CS31, and Y. enterocolitica fyuA and irp1 mutants, were cultured under iron-limiting conditions and the ability of the different strains as well as of purified yersiniabactin to induce the growth of the indicator strains was evaluated. The cross-feeding assays showed that Y. enterocolitica WA fyuA2, which produces yersiniabactin but lacks the yersiniabactin FyuA receptor, is able to cross-feed both P. damselae subsp. piscicida DI21 and CS31, whereas Y. enterocolitica WA-CS irp1 : : Kanr, which has the yersiniabactin FyuA receptor but is unable to synthesize yersiniabactin, is not (Table 4). The assays using purified yersiniabactin corroborated that both the parental P. damselae subsp. piscicida and the CS31 mutant are able to use this siderophore (Table 4).
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). This indicates that induction of growth in Yersinia strains by P. damselae subsp. piscicida is actually due to the production of a siderophore molecule whose synthesis is dependent on the irp gene cluster. Altogether, these results suggest that the siderophore produced by subsp. piscicida could be structurally and functionally related to yersiniabactin.
Distribution of irp cluster genes in P. damselae subsp. piscicida and subsp. damselae isolates
The presence of genes of the irp cluster described in this study in various P. damselae isolates of the two subspecies and with diverse isolation origins was tested by Southern hybridization. Chromosomal DNA samples were digested with HindIII, and Southern blotting was individually performed with probes internal to the dahP, araC1, frpA, irp2, irp1, irp9 and irp5 genes. For all the hybridization probes, the results were equivalent: all these genes were found to be absent in all the subsp. damselae strains and were present uniquely in subsp. piscicida strains DI21, B51, 666.1, PC554.2, PC435.1 and ATLIT 2 (Fig. 5). Interestingly, the two subsp. piscicida avirulent strains (EPOY 8803-II and ATCC 29690) tested negative for all these genes (Fig. 5). All the positive strains are recognized as virulent for fish (Table 1) and were isolated in Spain and Portugal, with the exception of ATLIT 2, which was isolated in Israel. When the primer pairs used to amplify the DNA probes were also used in a PCR-based screening with all the strains, the data for the presence of irp cluster genes were equivalent to those obtained by Southern blotting (data not shown).
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). This DNA region contains homologues of all the P. damselae subsp. piscicida genes, with the exception of dahP (Fig. 6). Although the P. profundum PBPRA1666 gene, which is annotated as a putative AMP-binding enzyme, does not have a gene counterpart in subsp. piscicida, it encodes a protein that is highly homologous to the C-terminus of P. damselae subsp. piscicida Irp1, suggesting that the subsp. piscicida Irp1 protein is actually the counterpart of the P. profundum proteins Irp1 and PBPRA1666. None of the published Vibrio genomes was shown to contain a gene cluster homologous to that described in this study (Heidelberg et al., 2000; Chen et al., 2003; Makino et al., 2003; Ruby et al., 2005). However, we identified genes similar to those of the P. damselae subsp. piscicida irp cluster in V. cholerae RC385 (GenBank ref. AAKH00000000), a strain whose genome project is being completed at TIGR (The Institute for Genomic Research, http://www.tigr.org/). Interestingly, these genes are distributed into two gene clusters separated by approximately 75 kb (Fig. 6).
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), and whereas the Yersinia HPI contains genes that are divergently transcribed, the P. damselae subsp. piscicida genes are all transcribed from the same DNA strand. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fouz et al., 1997). The genetic content can vary considerably between strains of the same species, and this variation is particularly frequent in genes that encode virulence factors. In this scenario, SSH has been successfully applied to the identification of genomic differences between bacterial strains (Winstanley, 2002), and it has been recently applied to the assessment of genetic diversity among P. damselae subsp. piscicida isolates (Juiz-Río et al., 2005b). In the present study, by using SSH, we have identified a subsp. piscicida-specific gene cluster spanning 35 kb, involved in siderophore biosynthesis. This cluster includes 11 putative ORFs that can be co-transcribed from a promoter upstream of dahP. However, we understand that this does not exclude the possibility that additional promoter sequences may exist upstream of other genes of this cluster.
The Fur protein is the major repressor of iron-uptake systems in Gram-negative bacteria, repressing transcription of the genes and thus limiting the entry of excess iron into the cell (Escolar et al., 1999). Two high molecular mass protein bands, which are predicted to correspond to Irp1 and Irp2 encoded within the irp cluster, are induced under conditions of iron limitation, which could be explained by the presence of Fur-binding sequences predicted upstream of dahP and araC1. In accordance with this, previous studies demonstrated that P. damselae subsp. piscicida DI21 harbours a fur homologue that can act as an iron-dependent transcriptional repressor (Juiz-Río et al., 2004; Osorio et al., 2004).
Genes involved in siderophore biosynthesis can also be subject to transcriptional activation. The AraC1 and AraC2 proteins described in this study are putative members of the AraC family of transcriptional activators, which are classified based on a conserved 99 aa stretch usually found within the C-terminus. This conserved region is predicted to encode two helix–turn–helix motifs that function in DNA binding (Gallegos et al., 1997). The homology of AraC1 and AraC2 with other proteins in databases was shown to be restricted to the C-terminus, indicating that the main variability resides within the N-terminus. Interestingly, there is increasing evidence that the N-terminus of AraC-family activators regulating siderophore synthesis and transport genes contains cofactor-binding regions, and this co-factor has proved to be the cognate ferri-siderophore complex (Tanabe et al., 2005; Michel et al., 2005). It is also interesting that AraC2 shows similarity to YbtA, a protein that controls expression of yersiniabactin synthesis genes within the Yersinia spp. HPI in a yersiniabactin-dependent manner (Fetherston et al., 1996; Anisimov et al., 2005). It is noteworthy that Yersinia irp1 mutants do not produce HMWP1 and HMWP2 (Pelludat et al., 1998), since yersiniabactin is required for upregulation of irp genes. In contrast, we have demonstrated here that a P. damselae subsp. piscicida irp1 mutant still produces irp2 (Fig. 2). These results suggest that differences in gene regulation exist between Yersinia and P. damselae subsp. piscicida gene clusters, and either AraC1, AraC2 or both could play a regulatory role distinct from YbtA. In this context, we could not find predicted YbtA-binding sites in the putative promoter regions of dahP and AraC2 (data not shown).
FrpA is the putative outer-membrane siderophore receptor of P. damselae subsp. piscicida DI21. It shows high similarity to the yersiniabactin receptor FyuA and to the quinolobactin receptor of Ps. fluorescens, but not to any of the siderophore receptors described in vibrios, suggesting that P. damselae subsp. piscicida might utilize a siderophore that is not related to any of the catecholate- or hydroxamate-type siderophores described to date in other vibrios.
The insertional inactivation of the irp cluster led to an impairment of growth under conditions of iron limitation, suggesting that this cluster encodes a siderophore-based mechanism that is crucial for iron uptake. The absence of reaction in the CAS agar plates of the CS31 mutant indicates that this is the only high-affinity iron-scavenging system present in P. damselae subsp. piscicida DI21. The irp1 mutant displayed reduced virulence in fish, demonstrating that the presence of this siderophore-mediated iron-acquisition mechanism contributes significantly to the virulence of P. damselae subsp. piscicida. The high LD50 exhibited by the mutant strain (2.6x107) could even be due to LPS toxicity, which would mean that the siderophore mutant could be avirulent. Mutants in the irp1 gene of extraintestinal pathogenic E. coli carrying HPI were also less virulent than the parental strain (Schubert et al., 2002). The connection between siderophore-mediated iron-acquisition mechanisms and bacterial virulence is well established (Faraldo-Gómez & Sanson, 2003), and recent studies have demonstrated the importance of siderophore production in the virulence of fish pathogens (Stork et al., 2004; Fernández et al., 2004).
Protein sequence comparisons suggested that the siderophore produced by P. damselae subsp. piscicida is synthesized by an NRPS-mediated mechanism similar to that described for yersiniabactin. Based on the in silico analysis, the as yet uncharacterized siderophore produced by P. damselae subsp. piscicida DI21 might contain at least one salicylate and two cysteine residues in its structure. The dahP -encoded DAHP synthase is probably involved in the synthesis of chorismate. This is an intermediate in the biosynthesis of aromatic compounds, some of which are precursors of aromatic carboxy and amino acids which are part of the structure of siderophores. In this context, a dahP homologue has been described as part of the gene cluster involved in siderophore anguibactin biosynthesis in V. anguillarum (Alice et al., 2005). Irp9 is the candidate enzyme to be involved in the synthesis of salicylate, based on its high similarity to the Y. enterocolitica Irp9. Irp3 and Irp4 are counterparts of the Yersinia spp. Irp3 (or YbtU) and Irp4 (or YbtT). Therefore, the P. damselae subsp. piscicida gene cluster described in this study contains all the gene counterparts of the yersiniabactin biosynthetic complex (Crosa & Walsh, 2002). All these data suggest that the DI21 siderophore might be structurally related to yersiniabactin (Drechsel et al., 1995), and this hypothesis is supported by the results obtained in the cross-feeding assays. However, the differences in the domain composition between the Yersinia and DI21 Irp1 and Irp2 homologues suggest that the two siderophores could have some differences in their structures.
The gene cluster described in this study was found to be absent in subsp. damselae strains, but was present in subsp. piscicida strains isolated from Spain and Portugal and one isolate from Israel. The two subsp. piscicida avirulent strains as well as all the isolates from Japan lacked this system, although they have been reported to produce siderophores (Magariños et al., 1994). This suggests that these strains harbour an as yet uncharacterized genetic system for siderophore production. In a previous study (Magariños et al., 2000), it was proposed that two distinct clonal lineages exist within P. damselae subsp. piscicida, represented by the European isolates on the one hand, and the Japanese isolates on the other hand. Our data support this hypothesis and indicate that DNA fragments from the operon described here could be used as genetic markers for the epidemiological typing of strains of this fish pathogen.
As P. damselae subsp. piscicida is a member of the Vibrionaceae, it is expected that the genes responsible for siderophore biosynthesis in this species would show high similarity to homologous genes in other vibrios. However, most of the predicted protein sequences encoded by this gene cluster did not show significant similarity to described siderophore synthesis and transport gene clusters of the Vibrionaceae. Homologous gene clusters exist in P. profundum SS9 and V. cholerae RC385, although their role in siderophore biosynthesis remains untested. It is noteworthy that the P. profundum irp homologues are located downstream of an asn-tRNA gene (Fig. 6), which suggests that this gene cluster may have been integrated in the chromosome in a similar way as described for the Yersinia HPI. Although we did not find evidence of asn-tRNA genes upstream of the P. damselae subsp. piscicida irp cluster, this does not exclude the possibility that this cluster is part of a laterally transmitted integrating element, since genetic rearrangements of the flanking sequences could have occurred after integration, or a mechanism of integration independent of the presence of tRNA genes could have taken place. In this context, no tRNA genes were found in the V. cholerae RC385 homologous cluster.
Among the gene clusters showing homology to the irp cluster of P. damselae subsp. piscicida, the Yersinia HPI is the most widely studied. The HPI is widespread among both pathogenic and non-pathogenic taxa of Enterobacteriaceae (Schubert et al., 1998; Bach et al., 2000; Olsson et al., 2003) and its ability to be horizontally transmitted has recently been demonstrated (Lesic & Carniel, 2005). The similarity between HPI genes and those described here in the irp cluster suggests that the P. damselae subsp. piscicida siderophore biosynthesis genes might have a common evolutionary origin with the Yersinia HPI. Recently, gene clusters similar to the Yersinia HPI have also been described in yersiniabactin-producing strains of Pseudomonas syringae and Photorhabdus luminescens (Bultreys et al., 2006), providing more clues about the evolution and dispersion of yersiniabactin-related genes.
The differential occurrence of this gene cluster among P. damselae strains, together with its low similarity to siderophore synthesis genes of other vibrios, suggest that the P. damselae subsp. piscicida siderophore biosynthesis gene cluster could have been acquired by horizontal transfer. Future studies will help to ascertain whether the irp cluster is part of a genomic island and if this DNA region can be mobilized in a similar way as that described for the HPI.
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REFERENCES |
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Received 7 June 2006;
revised 26 July 2006;
accepted 3 August 2006.
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), and CS31 mutant (
) in CM9 minimal medium supplemented with 10 µM FeSO4 (iron-sufficient conditions; continuous lines), and in CM9 in the presence of the iron chelator 2,2'-dipyridyl at a concentration of 60 µM (iron-limited conditions; discontinuous lines).
