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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ749789AJ749795, AJ749797AJ749804, AJ749806AJ749809, AJ749812, AJ870983AJ870986 and AJ888462AJ888463.
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INTRODUCTION |
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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.
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METHODS |
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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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RESULTS AND DISCUSSION |
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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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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 1520 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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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
-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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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.
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ACKNOWLEDGEMENTS |
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Received 13 January 2005;
revised 30 March 2005;
accepted 19 May 2005.
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