Subtractive hybridization reveals a high genetic diversity in the fish pathogen Photobacterium damselae subsp. piscicida: evidence of a SXT-like element

Sandra Juíz-Río1,2, Carlos R. Osorio1, Víctor de Lorenzo2 and Manuel L. Lemos1

1 Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
2 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain

Correspondence
Manuel L. Lemos
mlemos{at}usc.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Photobacterium damselae subsp. piscicida is the causative agent of fish pasteurellosis, a severe disease affecting cultured marine fish worldwide. In this study, suppression subtractive hybridization was used to identify DNA fragments present in the virulent strain PC554.2, but absent in the avirulent strain EPOY 8803-II. Twenty-one genomic regions of this type (that included twenty-six distinct putative ORFs) were analysed by DNA sequencing. Twenty ORFs encoded proteins with homology to proteins in other bacteria, including four homologues involved in siderophore biosynthesis, and four homologues related to mobile elements; three of these were putative transposases and one was a putative conjugative transposon related to the Vibrio cholerae SXT element. This sequence was shown to be integrated into a prfC gene homologue. Six ORFs showed no significant homology to known bacterial proteins. Among the 21 DNA fragments specific to strain PC554.2, 5 DNA fragments (representing 7 ORFs) were also absent in the avirulent strain ATCC 29690. The analysis of these differential regions, as well as the screening of their presence in a collection of strains, demonstrated the high genetic heterogeneity of this pathogen.


Abbreviations: RDA, representational difference analysis; SSH, suppression subtractive hybridization

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ749789–AJ749795, AJ749797–AJ749804, AJ749806–AJ749809, AJ749812, AJ870983–AJ870986 and AJ888462–AJ888463.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The marine Gram-negative bacterium Photobacterium damselae subsp. piscicida is the causative agent of fish pasteurellosis or pseudotuberculosis in warm water marine fish (Magariños et al., 1996a). This disease, which was first described in wild populations of white perch (Morone americanus) (Snieszko et al., 1964) and striped bass (Morone saxatilis) (Janssen & Surgalla, 1968), has affected wild and cultured marine fish in the USA and Japan (Kitao, 1993), and since 1990 in various European countries (Magariños et al., 1996a). The pathogenesis of P. damselae subsp. piscicida in fish is a multifactor process not yet fully understood. It is believed that the main virulence factors of this micro-organism consist of polysaccharide capsular material (Magariños et al., 1996b), and a high affinity siderophore-mediated iron-sequestering system (Magariños et al., 1994). Extracellular products, including a variety of enzymes, as well as adherence and invasive capacities, are also believed to play a role in virulence (reviewed by Romalde, 2002). However, as a whole, little is known about the molecular biology of this micro-organism or the molecular basis of the putative virulence factors. Although attempts to assess the genetic variability within P. damselae subsp. piscicida strains have been conducted using DNA fingerprinting techniques such as ribotyping (Magariños et al., 1997) and RAPD (random amplified polymorphic DNA) analysis (Magariños et al., 2000), most of these techniques proved to be of limited value in discriminating between strains. The limited amount of sequence data available, together with the finding that no serotypes can be differentiated within this pathogen, led to the consideration that the species was homogeneous (Magariños et al., 1996a).

Differences in virulence between isolates of the same bacterial species are related to differences in gene content and/or gene expression (Akopyants et al., 1998). Comparison of the genome sequences of non-pathogenic and pathogenic strains can provide useful information on genes that are specific for highly virulent isolates. Some of these differentially occurring genes may determine strain-specific characteristics such as virulence factors (Tinsley & Nassif, 1996; Groisman & Ochman, 1997; Reckseidler et al., 2001), which can be used as a valuable tool to establish the nature and severity of disease.

Representational difference analysis (RDA) (Lisitsyn, 1995; Tinsley & Nassif, 1996), which is based on suppression subtractive hybridization (SSH) (Akopyants et al., 1998; Diatchenko et al., 1996; Gurskaya et al., 1996), is a powerful technique for analysing the differences between two complex genomes, and for identifying DNA sequences that are present in one strain (the tester), but absent in another strain (the driver). This technique has been used in different bacterial species to identify genomic differences between virulent and avirulent strains (Calia et al., 1998; Mahairas et al., 1996; Zhang et al., 2000). Since pathogenesis is a multifactor process, SSH analysis can be useful for revealing genes that otherwise would not be detected by a genetic screen of random mutants. This is particularly advantageous for genetic studies with P. damselae subsp. piscicida, in which previous attempts to use transposon-based mutagenesis in our laboratory were unsuccessful. Furthermore, comparison of two complete genomes can give us a more complete genetic picture of the pathogen than other techniques.

Thus, our goal was to carry out a screen for P. damselae subsp. piscicida genes that are differentially present in a collection of virulent and avirulent strains. Implementing the SSH methodology, we have investigated genetic differences between two strains selected on the basis of their LD50. The analysis of tester specific regions, as well as their screening in other virulent and avirulent strains, demonstrated the high genetic heterogeneity of this pathogen. Evidence in the tester strain of a conjugative transposon related to Vibrio cholerae SXT element is also described.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, media and DNA extraction.
Strains used in this study are listed in Table 1. P. damselae subsp. piscicida strains were routinely grown at 25 °C in Tryptic Soy Agar (Difco) supplemented with 1 % NaCl (TSA-1). Escherichia coli strains were routinely grown at 37 °C in Luria broth (LB). When necessary, media were supplemented with ampicillin (100 µg ml–1). All strains were stored frozen at –80 °C in LB broth with 20 % (v/v) glycerol. Total genomic DNA from P. damselae strains was prepared as described by Ausubel et al. (1995). Plasmid DNA was purified and DNA was extracted from agarose gels using kits from Qiagen.


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Table 1. Bacterial strains

 
Suppression subtractive hybridization.
Bacterial genome subtraction was performed following the user manual of the PCR-Select Bacterial Genome Subtraction Kit (Clontech). Briefly, tester strain (PC554.2) and driver strain (EPOY 8803-II) genomic DNA (2 µg) were each digested with 10 units RsaI for 5 h. The tester DNA was then aliquoted into two tubes, and the DNA in each aliquot was ligated to a different adaptor provided with the kit (tester 1-1 and 2-1). Two hybridizations were carried out; in the first hybridization, an excess of driver was added to each adaptor-ligated tester sample, and samples were then heat denatured and allowed to anneal. In the second hybridization, the two samples from the first hybridization were mixed together, but 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) and an iCycler thermal cycler (Bio-Rad). The PCR products were cloned using a pGEM-T Easy TA cloning kit (Promega) and transformed into E. coli DH5{alpha}. Recombinant clones were screened by restriction analysis to allow duplicated clones to be discarded before proceeding to DNA sequencing.

DNA sequencing.
DNA sequences were determined by the dideoxy chain terminator method on plasmid products, using an Applied Biosystems Prism 3700 automated DNA sequencer and the dye termination method. The sequences were edited with BioEdit analysis software. The European Bioinformatics Institute EMBL database was screened with the FASTA3 and BLAST algorithms.

DNA hybridization and PCR.
For DNA hybridization, DNA inserts from some of the subtracted clones were used as probes to screen the genomic DNA of a collection of P. damselae subsp. piscicida strains. For dot-blot hybridization, chromosomal DNA samples were diluted in a final volume of 50 µl in TE buffer so that each sample contained approximately 2 µg DNA. Diluted samples were boiled for 5 min to denature dsDNA and immediately transferred on a nylon membrane (Boehringer Mannheim) previously activated with 2x SSC, using a Minifold II apparatus (Schleicher & Schuell). The transferred DNA was fixed to the membrane by exposure to UV light for 2 min in a ultraviolet cross-linker (Amersham). DNA probe labelling and hybridization were carried out using the ECL DNA labelling and detection system (Amersham), following the manufacturer's instructions.

Suitable oligonucleotides that flanked DNA inserts of clones pRDA5, 19, 23, 25 and 31 were designed and used in a PCR-based screening of a collection of P. damselae subsp. piscicida strains. When a PCR product of the expected size was amplified, the result was scored as positive for the presence of that gene.

To assess the attP attachment site of the putative SXT-like element into a chromosomal attachment site, attB, genomic DNA of strain PC554.2 was cut with BamHI and ligated with pUC118 plasmid that had been similarly digested. The ligation reactions were used as the DNA template in PCR reactions with M13fw primer (targeted to the pUC118 polylinker) and either primer P4 or P5, which are targeted to the left (attL) and right (attR) junctions of the putative SXT-like element, respectively (Hochhut & Waldor, 1999). The resulting PCR products were cloned into pGEM-T Easy (Promega) and sequenced. This strategy allows the amplification of DNA regions in the vicinity of the insertion site of an SXT-like element.

Nucleotide sequence accession numbers.
The DNA sequences described in this article have been assigned the EMBL accession numbers listed in Tables 2 and 3.


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Table 2. Summary of the sequence analysis of clones inserts specific for P. damselae subsp. piscicida pathogenic strain PC554.2, and absent from avirulent strain EPOY 8803-II

 

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Table 3. PCR amplification of SXT-like sequences in the P. damselae subsp. piscicida tester strain PC554.2, and nucleotide sequence comparison to homologous sequences in SXT-like conjugative and integrative elements in other {gamma}-proteobacteria

 

   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
In the last few years, several strategies have been developed for the identification of bacterial genes essential for infection, such as the use of in vivo expression technology (Mahan et al., 1993), signature-tagged transposon mutagenesis (Shea et al., 1996) and microarray DNA chips (de Saizieu et al., 1998). In this study, we used SSH to identify genetic differences between two P. damselae subsp. piscicida strains, one highly pathogenic and the other innocuous, and isolated twenty-one distinct types of clone that were tester specific. These results provide a more complete picture of the genetic background of P. damselae subsp. piscicida, whose genome has not yet been sequenced.

Genomic subtraction between P. damselae subsp. piscicida PC554.2 and EPOY 8803-II
For the SSH procedure we used as the tester the highly virulent strain PC554.2 isolated from sole (Solea senegalensis) (Magariños et al., 2003), and as the driver the strain EPOY 8803-II, isolated from Epinephelus akaara (Magariños et al., 1992), which displays a much lower degree of virulence. The sizes of subtracted fragments were between 0·7 and 2 kb. Ninety-eight clones were obtained after two rounds of hybridization. Ten clones were found to have no inserts, and sixty-four duplicated clones were discarded on the basis of their restriction pattern. The remaining 23 clones were examined by dot-blot hybridization analysis to check the specificity of the technique. In addition to the tester and driver strains, one virulent (DI21) and one avirulent strain (ATCC 29690) were included in the dot-blot screening. Only 2 clones proved to be false positives (pRDA15 and pRDA36), while the other 21 hybridized with the tester genome but not with the driver genome (Fig. 1). Four clones were present only in the two virulent strains, whereas sixteen clones were found in the two virulent strains plus in the avirulent strain ATCC 29690. One clone (pRDA5) was specific to the tester strain PC554.2 (Fig. 1).



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Fig. 1. Dot-blots of subtracted fragments of chromosomal DNA from P. damselae subsp. piscicida DI21 (a), PC554.2 (tester strain) (b), EPOY 8803-II (driver strain) (c) and ATCC 29690 (d). The DNA subtracted fragments used as labelled probes are indicated on the left of the panels.

 
The selected 21 subtracted fragments were sequenced and a homology search was carried out (Table 2). A total of 26 distinct putative ORFs (either partial or complete) were inferred, among which 6 (clones pRDA13, pRDA14, pRDA17, pRDA24, pRDA32 and pRDA37) showed no significant matches with entries in the databases. These proteins are thus referred to as putative P. damselae subsp. piscicida proteins. To date, more than 100 bacterial genomes, including those of several important pathogens, have been sequenced, revealing that around 25 % of the ORFs are hypothetical genes without known function (Fraser, 2000).

The remaining 20 predicted ORFs showed homology to proteins described in other bacterial species. Clones into this category were divided into two subgroups. In subgroup A we included the ORFs that were found to correspond to putative insertion sequences elements of P. damselae subsp. piscicida, and in subgroup B we included the remaining RDA clones.

Presence of transposase genes in P. damselae subsp. piscicida
SSH has been successfully used to detect the differential presence of insertion sequences in other bacteria (Lai et al., 2000; Sawada et al., 1999; DeShazer, 2004). The putative insertion sequences detected in P. damselae subsp. piscicida included three different transposases, with homology to Vibrio vulnificus putative transposases A and B, and to a Shigella flexneri transposase (Table 2). Several clones contained a homologue of the V. vulnificus transposase A. It is noteworthy that the DNA sequence flanking the transposase gene was different in each clone, indicating that this gene occurs in a multicopy fashion in the genome of the tester strain. A single clone (pRDA16) contained a homologue of the V. vulnificus transposase B. Interestingly, when this transposase B gene was used as a probe in a Southern blot of chromosomal DNA cut with BglII and PstI, it was also shown to occur in a multicopy fashion (about 15–20 copies) in the tester strain, as well as in three additional P. damselae strains, while it proved to be absent in the driver strain (Fig. 2). The occurrence of multiple copies of a sequence in a genome seems to be a feature typical of small insertion sequences (Schneider & Blot, 2003). In addition, clone pRDA37 contained an ORF with homology to a S. flexneri transposase found in the virulence plasmid pCP301 (Jin et al., 2002).



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Fig. 2. Southern blot of chromosomal DNA doubly digested with BglII and PstI, hybridized with the transposase B gene from clone pRDA16. Lanes indicate P. damselae subsp. piscicida strains: (a) PC554.2 (tester), (b) DI21, (c) ATCC 29690, (d) B52, (e) EPOY 8803-II (driver). Molecular sizes (kb) are denoted on the left.

 
These putative transposase genes are believed to be the first identified in P. damselae subsp. piscicida to date. In all cases, the putative transposase genes were inserted so that they interrupted ORFs. The identification of transposase elements or markers specific to individual strains or clones, has been shown to be an important starting point in the identification of genomic islands implicated in virulence (Winstanley, 2002). It has been reported that the typing of closely related strains of the same species can be accomplished by studying the variation in mobile genetic elements (Lawrence et al., 1989). The presence of transposases and their distribution along the genome could be an adequate method for typing P. damselae subsp. piscicida strains, in which other typing methods proved to be of limited value (Magariños et al., 1997; 2000).

Identification of SXT-like element DNA sequences in the tester strain
Within the subgroup A clones we also included clone pRDA5, which contained a partial ORF with high similarity to an uncharacterized protein included within the SXT element of V. cholerae. The identity at the nucleotide sequence level between pRDA5 and this V. cholerae homologue was as high as 94 %. This element is a representative of a family of conjugative transposon-like mobile genetic elements that encode multiple antibiotic-resistance genes (Beaber et al., 2002). That ORF also showed homology to a hypothetical protein of Providencia rettgeri found in the conjugative element R391 (Böltner et al., 2002). R391 and its relatives carry specific phenotypes that are also found in genomic islands (including symbiosis and pathogenicity islands). Both R391 and the SXT element share functional and structural similarities to genomic islands, conjugative transposons and bacteriophages.

To further test the hypothesis that a SXT-like conjugative element exists in the genome of the tester strain, PCR amplification and sequence analysis were employed to help identify DNA sequences that are well-conserved among SXT-like elements described in several species. These elements share a conserved ‘backbone’ that includes an integrase gene and other genes related to excision and integration, conjugative transfer and regulation (Beaber et al., 2002). A recent study described how primer pairs targeted to conserved DNA stretches can be useful for detecting DNA sequences of SXT-like elements in {gamma}-proteobacteria (Böltner and Osborn, 2004).

We selected five primer pairs (Table 3) targeted to genes encoding an integrase (int), a relaxase (traI), a putative pilus assembly protein (traC) and a putative pilus assembly and synthesis protein (traN). These primers proved to be effective for amplifying the sequences of V. cholerae SXT, P. rettgeri R391, Proteus mirabilis R997 and Shewanella putrefaciens pMERPH elements (Böltner and Osborn, 2004). The PCR amplifications yielded products of the size expected based on the DNA sequence of the P. rettgeri conjugative element R391 (Böltner et al., 2002) (Fig. 3a). DNA sequencing of the five amplicons in the tester strain demonstrated that SXT-like sequences exist in the genome of P. damselae PC554.2, and showed a high percentage of sequence identity to homologous sequences described in SXT-like elements (Table 3). The same primers were employed with DNA of strains EPOY 8803-II, ATCC29690 and DI21, but no amplification was obtained (data not shown), which is in accordance with the dot-blot hybridization results with pRDA5, and suggests that the SXT-like sequences are exclusive to strain PC554.2. When we carried out hybridization experiments with 29 strains of P. damselae subsp. piscicida using pRDA5 insert DNA as a probe, we found that it is present uniquely in the tester strain PC554.2 (Table 4).



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Fig. 3. (a) Amplification of genes of the putative SXT-like conjugative element in P. damselae subsp. piscicida strain PC554.2. A scheme of the amplified genes is shown above the agarose gel of the PCR products generated with the primers listed in Table 3. Open white arrows denote ORFs of the P. rettgeri R391 conjugative transposon, and small black arrows represent the relative location of primers. attL and attR denote the putative left and right junctions of the SXT-like element in the bacterial chromosome, respectively. The prfC gene is denoted as a hatched arrow located downstream of the attR junction. Lanes M1 and M2 contain 1 kb and 100 bp molecular size markers, respectively. The lanes and the predicted product sizes are as follows: lane 1, complete int gene and flanking bases (1427 bp); lane 2, int gene internal (1034 bp); lane 3 traI gene internal (1008 bp); lane 4, traC gene internal (553 bp); lane 5 traN gene internal (1044 bp). (b) Nucleotide sequence alignment of P. damselae subsp. piscicida PC554.2 and V. cholerae O139 chromosomes at the left (top) and right (bottom) SXT-like chromosome junctions. The 17 bp duplication resulting from integration of the SXT-like element, and which constitutes the core of att sites, is shown in bold and underlined. The nucleotide sequences of primers P4 and P5 are shown in bold and italics. The alternative ATG start codon of the 5' terminus of prfC gene, replacing the chromosomal 5' end and restoring a functional prfC gene upon chromosomal integration, is shown in bold and boxed. Numbers indicate positions in the SXT sequence of V. cholerae (accession no. AY055428).

 

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Table 4. Distribution of tester-specific DNA fragments among virulent and avirulent strains of P. damselae subsp. piscicida

Results were obtained by dot-blot hybridization and PCR.

 
Chromosomal integration of SXT-like conjugative transposons occurs via site-specific recombination between a 17 bp sequence found in the SXT element and a similar 17 bp sequence in the prfC gene that encodes the peptide chain release factor 3 (Hochhut & Waldor, 1999; McGrath & Pembroke, 2004). Recombination between the chromosomal attB and the SXT attP generates the left and right junctions attL and attR, respectively. The integration site in the chromosome of strain PC554.2 was assessed using primers targeted to the left and right junctions of this element within the bacterial chromosome (Hochhut & Waldor, 1999; Ahmed et al., 2005). PCR products were obtained that included the attL and attR junctions as well as neighbour chromosomal DNA. Sequencing of the amplicons revealed nucleotide sequences highly similar to those described for the attL and attR sites of V. cholerae SXT element and related conjugative transposons (Table 3), and also demonstrated that these sequences are inserted within a prfC homologue in strain PC554.2 (Fig. 3b). The SXT-like sequence disrupted prfC, but encoded a novel 5' sequence that presumably restores the function of the gene (Fig. 3b). A 17 bp region conforming the putative att core sequence was also encountered at the attP site (Fig. 3b). These results demonstrate that sequences nearly identical to the attL and attR sites of the attP attachment site of the V. cholerae SXT element are present in the chromosome of P. damselae subsp. piscicida PC554.2, and that the attR junction reconstitutes a complete copy of the prfC gene.

Altogether, these results strongly suggest that a SXT-like integrative and conjugative element is present in the genome of P. damselae subsp. piscicida PC554.2. To date, SXT-like elements have been described in as few as six bacterial species (McGrath & Pembroke., 2004; Ahmed et al., 2005). The high similarity between the SXT-related sequences analysed in our study and those reported previously supports the hypothesis that these types of elements share a common ancestral origin (Beaber et al., 2002; Böltner and Osborn, 2004). The fact that these sequences were found in only one recently isolated strain (Magariños et al., 2003), as well as their high homology to V. cholerae SXT sequences, suggests that a possible recent horizontal transfer of these sequences has occurred.

Clones containing ORFs homologous to those in other bacteria
The subgroup B comprises the remaining RDA clones that are summarized in Table 2. Among them, clones were isolated that contained ORFs encoding proteins implicated in the transport of molecules across the bacterial membrane (pRDA2, pRDA7, pRDA8 and pRDA38), as well as putative proteins involved in protein secretion.

Clones pRDA23, pRDA31, pRDA33 and pRDA34 contained putative members of iron-sequestering systems based on siderophore production. An ORF in pRDA31 showed homology to the vibD gene, required for late steps of vibriobactin biosynthesis in pathogenic strains of V. cholerae (Wyckoff et al., 2001). Similarly, pRDA33 and pRDA34 harboured ORFs with homology to two different putative chorismate mutases, enzymes also involved in the synthesis of siderophores. Clones pRDA31 and pRDA23 contained homologues of genes described as part of the virulence plasmid pJM1 of Vibrio anguillarum (Di Lorenzo et al., 2003) that encodes an iron-sequestering system. Homologues include a phosphopantetheinyl transferase (clone pRDA31), an enzyme that could be involved in the synthesis of the siderophore anguibactin, and a putative DAHP (3-deoxy-D-arabinoheptulosonate-7-phosphate) synthase in clone pRDA23. This clone also contained an ORF with homology to a putative member of the AraC family of transcriptional activators. Members of this family act as positive regulators of genes encoding siderophore receptors and for enzymes involved in siderophore biosynthesis, as is the case of Yersinia pestis YbtA (Fetherston et al., 1996). This ORF in clone pRDA23 has been previously isolated in P. damselae subsp. piscicida strain DI21, as part of a gene cluster that included a putative ferrisiderophore receptor and additional iron-regulated genes (Osorio et al., 2004).

Clone pRDA25 includes two ORFs homologous to the SecD and SecF proteins of the E. coli Sec locus, which comprises nine genes involved in protein translocation across the membrane (Manting & Driessen, 2000). It has been suggested that SecD and SecF proteins form a complex in the inner membrane that functions at the late steps of protein export (Economou et al., 1995). The presence of this protein export system could be related to virulence in P. damselae subsp. piscicida isolates, since extracellular proteins considered as potential virulence factors need to be translocated across the bacterial envelope through a protein export system.

An ORF with homology to a Srmb helicase of Photobacterium profundum has also been isolated (clone pRDA4). It is not clear whether the differential presence of helicase genes can be related to virulence. Curiously, using SSH, two different helicase genes were reported to be present in a pathogenic Pseudomonas aeruginosa isolate and absent in the driver avirulent strain (Choi et al., 2002).

Occurrence of the isolated sequences in a collection of P. damselae subsp. piscicida strains
We focused our attention on clones pRDA19, pRDA23 and pRDA31, representative ORFs present in the two pathogenic strains PC554.2 and DI21, but absent in the two non-pathogenic strains EPOY 8803-II and ATCC29690. We also included clone pRDA5 since it was present uniquely in the tester strain, and clone pRDA25 containing homologues to genes involved in protein translocation across the membrane in E. coli (Table 2). In order to survey the distribution of these DNA fragments, dot-blot hybridization was carried out with 9 virulent and 2 avirulent strains of P. damselae subsp. piscicida, as well as with 17 additional strains isolated from diseased fish but whose LD50 had not been determined. Results showed that a high degree of genetic heterogeneity exists in P. damselae subsp. piscicida isolates at the assayed DNA regions (Table 4). The same results were obtained when oligonucleotides specific for each of pRDA5, pRDA19, pRDA23, pRDA25 and pRDA31 inserts were used in a PCR-based screening (Table 4).

The dot-blot hybridization and PCR revealed a possible relationship between the presence of genes encoding elements of high affinity iron-transport systems and virulence. According to these experiments, they were absent in the two non-pathogenic isolates tested (Table 4), although they showed variable occurrence among other pathogenic isolates. Strains of P. damselae subsp. piscicida produce siderophores under iron-limiting conditions (Magariños et al., 1994), but little is known about the genetic basis of these iron-sequestering systems. The genes isolated in this study will help to provide a deeper insight into the iron-acquisition mechanisms of this fish pathogen.

Clone pRDA19 contained two partial ORFs with homology to two distinct, chromosomally linked cytochrome C oxidases. These ORFs also showed a differential occurrence among P. damselae subsp. piscicida isolates, being absent not only in the two avirulent strains but also in several virulent ones (Table 4).

The results obtained from the distribution of some of the genes isolated in the present study, in 28 virulent and avirulent strains of P. damselae subsp. piscicida, strongly suggest that the population of this fish pathogen is highly heterogeneous. None of the strains tested, other than PC554.2, harboured all the sequences described here, and none of the sequences were present in all the virulent isolates tested. This heterogeneity is also very evident among strains isolated from the same host and from the same geographical origin. As an example, each of the seven strains isolated from sole in Spain (Table 1) yielded a completely different gene content profile in the Southern blot and PCR screening, summarized in Table 4, data that clearly indicate the high genetic heterogeneity of the populations of this fish pathogen. We propose that some of the DNA sequences described in this study could serve as genetic markers for epidemiological typing of P. damselae subsp. piscicida strains.

Conclusion
Our results demonstrate that SSH is a successful technique for identifying genetic differences between virulent and avirulent P. damselae subsp. piscicida isolates. This is expected to provide insights into the virulence mechanisms of this fish pathogen. Although from the results described here a direct relationship between a particular sequence and virulence cannot be inferred (only two strains clearly considered as avirulent are available), the distribution of a particular gene in a bacterial population can provide clues on its implication in virulence, and clearly some of the sequences described here could be involved in the virulence of P. damselae subsp. piscicida. Isolation of genes related to iron-acquisition mechanisms as part of the subtracted sequences suggests a likely role of these systems in virulence, as has been demonstrated in other bacteria. In addition, the presence of several genes related to mobile elements among the subtracted fragments points to the presence of pathogenicity islands in P. damselae subsp. piscicida. Studies on this possibility are currently under way.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grant AGL2003-00086 from the Ministry of Science and Technology of Spain, and grant PGIDIT04PXIC23501PN from Xunta de Galicia to M. L. L. S. J. R also thanks the Spanish Ministry of Science and Technology for a predoctoral fellowship.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Ahmed, A. M., Shinoda, S. & Shimamoto, T. (2005). A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis. FEMS Microbiol Lett 242, 241–247.[CrossRef][Medline]

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Received 13 January 2005; revised 30 March 2005; accepted 19 May 2005.



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