The Vibrio seventh pandemic island-II is a 26·9 kb genomic island present in Vibrio cholerae El Tor and O139 serogroup isolates that shows homology to a 43·4 kb genomic island in V. vulnificus

Yvonne A. O'Shea, Shirley Finnan, F. Jerry Reen, John P. Morrissey, Fergal O'Gara and E. Fidelma Boyd

Department of Microbiology, UCC, National University of Ireland, Cork, Ireland

Correspondence
E. Fidelma Boyd
f.boyd{at}ucc.ie


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Vibrio cholerae is the aetiological agent of the deadly diarrhoeal disease cholera. In this study the 7·5 kb Vibrio seventh pandemic island-II (VSP-II) that is unique to V. cholerae El Tor and O139 serogroups was analysed and it was found to be part of a novel 26·9 kb genomic island (GEI) encompassing VC0490–VC0516. The low-GC-content VSP-II encompassed 24 predicted ORFs, including DNA repair and methyl-accepting chemotaxis proteins, a group of hypothetical proteins and a bacteriophage-like integrase adjacent to a tRNA gene. Interestingly, V. cholerae ORFs VC0493–VC0498, VC0504–VC0510 and VC0516, which encodes an integrase, were homologous to Vibrio vulnificus strain YJ016 ORFs VV0510–VV0516, VV0518–VV0525 and VV0560, which also encodes an integrase, respectively. Some ORFs showed amino acid identities greater than 90 % between the two species in these regions. In V. vulnificus strain YJ016, a 43·4 kb low-GC-content (43 %) GEI encompassing ORFs VV0509–VV0560 was identified and named V. vulnificus island-I (VVI-I). The 52 ORFs of VVI-I included a phosphotransferase system gene cluster, genes required for sugar metabolism and transposase genes. There was synteny and homology between the 5' region of V. cholerae VSP-II and the 5' region of V. vulnificus VVI-I; however, VVI-I contained an additional 31·5 kb of DNA between VV0526 and VV0560 in strain YJ016. A second V. vulnificus strain, CMCP6, did not contain the 43·4 kb VVI-I; in this strain two ORFs were found between the 5' and 3' flanking genes VV10636 and VV10632, showing 100 % identity to VV0508 and VV0561, respectively, which flank VVI-I.


Abbreviations: GEI, genomic island; VPI, Vibrio pathogenicity island; VSP-II, Vibrio seventh pandemic island-II; VVI-I, Vibrio vulnificus island-I


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Vibrio, which belongs to the Gram-negative {gamma}-Proteobacteria, is ubiquitous in marine and estuarine environments. There are 71 recognized species within the genus Vibrio, of which 12 species are pathogenic to humans, in the current List of Bacterial Names with Standing in Nomenclature (Euzéby, 1997; Farmer et al., 2003). Vibrio cholerae is the most important human pathogen belonging to the genus since it is the aetiological agent of the deadly diarrhoeal disease cholera. Cholera is endemic in many areas of the world, particularly those areas where clean drinking water is absent. V. cholerae pathogenic isolates contain two main virulence factors, cholera toxin (CT) and toxin co-regulated pilus (TCP), both of which help facilitate cholera spread and pathogenesis (Sears & Kaper, 1996; Taylor et al., 1987). CT, the main cause of the explosive diarrhoeal disease, is encoded on a filamentous bacteriophage CTX{pi} (Waldor & Mekalanos, 1996). The receptor for CTX{pi} on the V. cholerae cell, TCP, which is an essential intestinal colonization factor, is also encoded on a mobile genetic element named the Vibrio pathogenicity island (VPI) (Waldor & Mekalanos, 1996; Kovach et al., 1996; Karaolis et al., 1998; Taylor et al., 1987). To date only two V. cholerae serogroups, O1 and O139, are responsible for epidemic and pandemic cholera (Kaper et al., 1995). The O1 serogroup is divided into two biotypes, classical and El Tor. Of the seven recorded pandemics of cholera, the classical biotype was responsible for the sixth pandemic and the El Tor biotype is responsible for the seventh and ongoing pandemic, which began in 1961 (Kaper et al., 1995). The V. cholerae O139 serogroup emerged in 1992 as a major cause of epidemic cholera and is related to O1 serogroup strains (Albert et al., 1993; Cholera Working Group, 1993; Waldor & Mekalanos, 1994). Several studies have shown that O139 serogroup strains arose by the acquisition of the O139 biosynthesis genes by an O1 El Tor biotype strain, which was probably mediated by a bacteriophage (Berche et al., 1994; Bik et al., 1995, 1996; Waldor & Mekalanos, 1994; Stroeher et al., 1997).

The complete genome sequence of V. cholerae strain N16961 is available and has been shown to contain two circular chromosomes of 2·96 and 1·07 Mb (Trucksis et al., 1998; Heidelberg et al., 2000). Comparative genomic analysis of V. cholerae using a DNA microarray that displayed over 93 % of the predicted genes of strain N16961, determined differences in gene content between sixth (classical biotype) and seventh (El Tor biotype) pandemic isolates (Dziejman et al., 2002). These authors identified two genomic regions designated the Vibrio seventh pandemic island-I (VSP-I) and VSP-II that were unique to seventh pandemic El Tor isolates (Dziejman et al., 2002). VSP-I and VSP-II showed several characteristics of pathogenicity islands. VSP-I spans a 16 kb region (VC0175–VC0185) encompassing 11 ORFs, which include a deoxycytidylate deaminase-related protein, a transcriptional regulator, a patatin-related protein, a transposase and a number of hypothetical proteins, with a GC content of 40 mol%, in contrast to 47 mol% for the entire genome (Dziejman et al., 2002). The 7·5 kb VSP-II region encompasses eight ORFs (VC0490–VC0497), which encode a transcriptional regulator and a ribonuclease H1; however, the chromosomal boundaries of the region were not clearly defined (Dziejman et al., 2002).

Vibrio vulnificus is a human pathogen that is highly invasive, causing fulminate pulmonary septicaemia, with mortality rates as high as 60 %, one of the highest death rates of any food-borne disease (Linkous & Oliver, 1999; Strom & Paranjpye, 2000). In addition to septicaemia, V. vulnificus can also cause wound infections and gastroenteritis (Linkous & Oliver, 1999; Strom & Paranjpye, 2000). Similar to V. cholerae, V. vulnificus is associated with the estuarine environment and occurs in high numbers in molluscan shellfish (DePaola et al., 1994; Kaysner et al., 1987; Oliver et al., 1982; Chiavelli et al., 2001, Huq et al., 1984, Hood & Winter, 1997, Montanari et al., 1999). However, phylogenetic analysis based on 16S rRNA and hsp60 gene sequences indicate that V. vulnificus is more closely related to Vibrio parahaemolyticus than to V. cholerae (Kwok et al., 2002). A number of potential virulence factors have been identified in V. vulnificus clinical isolates, but these are also found associated with oyster isolates (DePaola et al., 2003). The V. vulnificus strain YJ016 genome sequence has been published recently (Chen et al., 2003). V. vulnificus strain YJ016 is a biotype 1 hospital isolate from Taiwan. The genome is 5·3 Mb, consisting of two circular chromosomes of 3·4 Mb (3262 ORFs) and 1·9 Mb (1697 ORFs), respectively, and a 49 kb plasmid (Chen et al., 2003). Of the 4959 genes identified, 1688 (34 %) encode hypothetical proteins, which accounts for most of the genes that are unique to the V. vulnificus genome.

In this paper, we show that the 7·5 kb VSP-II region (VCO490–VC0497) encompasses a 26·9 kb region (VC0490–VC0516) in V. cholerae biotype El Tor and O139 serogroup isolates.

The 5' region of V. cholerae VSP-II, ORFs VC0493–VC0498 and VC0504–VC0510, shows homology to a region in V. vulnificus strain YJ016, ORFs VV0510–VV0516 and VV0518–VV0525, respectively. We determine that in V. vulnificus strain YJ016, ORFs VV0510–VV0525 are part of a 43·4 kb genomic island (GEI) (VV0509–VV0560) named Vibrio vulnificus island-I (VVI-I) which encompasses a number of transport and sugar metabolism genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial isolates.
A total of 21 V. cholerae isolates were examined in this study (Table 1). The 21 V. cholerae isolates belonged to 4 different serogroups; 17 isolates belonged to serogroup O1, of which 2 isolates were of the classical biotype and 15 isolates were of the El Tor biotype. The O139 serogroup was represented by two isolates, the O37 serogroup by one isolate and the O141 serogroup by one isolate. Of the 21 strains examined, 15 were clinical isolates and 2 were environmental isolates (Table 1), and 11 contained the 7·5 kb VSP-II region (Table 1) (Dziejman et al., 2002; O'Shea et al., 2004). All Vibrio strains were grown in Luria–Bertani broth (LB) and stored at –70 °C in LB broth with 20 % (v/v) glycerol.


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Table 1. Strains used in this study

 
DNA isolation.
Chromosomal DNA was extracted from each V. cholerae isolate using the G-nome DNA isolation kit from Bio 101. Briefly, a single colony of each isolate was inoculated into 3 ml LB broth and incubated overnight at 37 °C with shaking at 150 r.p.m. The bacterial cells were pelleted at 3000 r.p.m. for 5 min, supernatant was discarded and the pellet was brought to a final volume of 1·85 ml in cell suspension solution. The cells were lysed and treated with RNase and protease. DNA was extracted with ethanol and resuspended in TE buffer.

PCR analysis.
PCR was used to assay 21 V. cholerae isolates for the presence of VSP-II. PCR analysis was carried out using 10 primer pairs (Table 2), the location of which can be seen in Fig. 1. Gene fragments were amplified from chromosomal DNA isolated from the 21 V. cholerae strains. PCR was performed in a 20 µl reaction mixture by using the following cycles: an initial denaturation step at 96 °C for 1 min followed by 30 cycles of denaturation at 94 °C for 30 s, 30 s of primer annealing at (49·6–58 °C) and 1–4 min of primer extension at 72 °C.


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Table 2. PCR primers used in this study encompassing VC0489–VC0517

 


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Fig. 1. (a) Regional variation in the mean proportion of GC content of the VSP-II region from V. cholerae N16961 based on a sliding window of 500 bp for V. cholerae. (b) Schematic representation of the organization of the VSP-II region in V. cholerae N16961. The position and direction of transcription of the ORFs are indicated by the direction of the open arrows. The black arrowheads above ORFs indicate the position of the PCR primers used in this study. The black bold horizontal lines indicate the position of the DNA probes used in Southern hybridization analyses in this study. The numbers refer to the genetic organization of the genes along the genome (Heidelberg et al., 2000). Genes are pattern-coded according to their function.

 
Southern hybridization analysis.
To confirm negative PCR results, Southern hybridization analysis was carried out with four DNA probes (490, 493, 502, 514) generated by PCR using the reference V. cholerae strain N16961 as template (Fig. 1). The 2·3, 3·6, 0·5 and 2·1 kb PCR products were purified using the ConceRT-PCR purification kit (Gibco-BRL) and 100 ng probe DNA was conjugated to horseradish peroxidase using the ECL direct nucleic acid labelling system (Amersham Pharmacia Biotech). DNA from each strain of interest was digested with the restriction enzyme EcoRI (Roche) and separated by electrophoresis in 0·6 % (w/v) 1x TBE agarose. Separated DNA fragments were transferred to a nitrocellulose membrane for Southern hybridization. Southern hybridization was carried out using the ECL direct nucleic acid labelling and detection system according to manufacturer's instructions (Amersham Pharmacia Biotech).

Nucleotide sequence and bioinformatic analysis.
A region spanning 26·9 kb from position 523 156 to 550 021 of the V. cholerae genome from strain N16961 and a 43·4 kb region spanning 498 755 to 542 138 of the V. vulnificus genome of strain YJ016 was analysed for sequence similarities using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/COG/). Bioinformatic analysis was performed using the software Artemis (Rutherford et al., 2000).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A 7·5 kb V. cholerae island VSP-II (ORFs VC0490–VC0497) that was confined to V. cholerae El Tor isolates and O139 serogroup isolates was described by Dziejman et al. (2002). Two genes 18·2 kb downstream of VSP-II, VC0514 and VC0516, were also identified as being present only in seventh pandemic El Tor O1 and O139 strains (Dziejman et al., 2002). Interestingly, ORF VC0516 encodes a bacteriophage-like integrase and was inserted adjacent to a tRNA gene (Fig. 1). To determine whether the original VSP-II region ORFs VC0490–VC0497 were linked to VC0516, we carried out PCR analysis with a set of 10 primer pairs that spanned the entire region from VC0489 (on the core chromosome), which marked the 5' flanking region, to VC0517 (on the core chromosome), which marked the 3' flanking region (Fig. 1 and Table 2). First, we investigated whether the region directly downstream of VC0497 was present in a range of V. cholerae El Tor VSP-II-positive and -negative strains (Dziejman et al., 2002; O'Shea et al., 2004). With primer pair VC0502F/VC0502R V. cholerae strains C5, 3038, F1873, F1875, F1939, 2125-98, GP33 and N16961, which are VPI-II-positive, gave an expected 0·5 kb PCR band (Fig. 2). The remaining six V. cholerae isolates, which are VSP-II-negative, gave no PCR product (Fig. 2). Next, we examined the distribution of VC0516 (integrase) among V. cholerae VSP-II-positive and -negative O1 and non-O1 serogroup isolates by PCR analysis with primer pair VC0516F/VC0516R. All four of the VSP-II-positive V. cholerae El Tor and O139 strains (N16961, SM115, MO2 and MO10) tested gave an expected 965 bp band, indicating the presence of VC0516 in these strains. In contrast, PCR analysis of five V. cholerae VSP-II-negative strains (569B, O395, V46, V52 and 468-83) failed to amplify a PCR band. Thus, both VC0502 and VC0516 only appear to be associated with strains that contained the previously described 7·5 kb VSP-II region (VC0490–VC0497).



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Fig. 2. PCR analysis of gene VC0502, which encodes a type IV pilin on the VSP-II island in biotype El Tor V. cholerae isolates. Lanes: 1, 1 kb marker; 2–14, V. cholerae strains RV79, C5, 2740-80, 468-83, 3038, F1873, F1875, F1939, 2164-78, 2125-98, GP33, GP43, GP155; 15, negative control; 16, positive control.

 
To determine whether the region between VC0497 and VC0516 was confined to VSP-II-positive isolates and absent amongst VSP-II-negative V. cholerae isolates, we carried out PCR analysis with four primer pairs that spanned this region (Table 2, Fig. 1, and Fig. 3). Primer pair VC0498F/VC0498R gave an expected 4·1 kb PCR product band from the four VSP-II-positive V. cholerae isolates (N16961, SM115, MO2 and MO10), whereas no product was amplified from the five V. cholerae VSP-II-negative isolates (569B, O395, V46, V52 and 468-83) tested. Similarly, PCR primer pairs VC0504F/VC0504R, VC0512F/VC0512R and VC0514F/VC0514R gave expected 3·2, 3·9 and 2·1 kb PCR product bands with the four VSP-II-positive V. cholerae isolates and, as expected, the five VSP-II-negative strains tested gave no PCR product (Fig. 3). The absence of the 19·4 kb region between VC0497 and VC0516 in VSP-II-negative V. cholerae isolates was verified by Southern hybridization analysis using two DNA probes (502, 514) derived from PCR fragments generated using primer pairs from Table 2 which spanned the region of interest (Fig. 1). No hybridization fragments were obtained with the DNA probes for 11 V. cholerae VSP-II-negative strains and, as expected, a positive hybridization band was obtained for V. cholerae VSP-II-positive strains, which contain this region (Fig. 4 and data not shown).



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Fig. 3. PCR analysis of VSP-II in V. cholerae O1 and non-O1 serogroup isolates. (a) Gene VC0489, which indicates the 5' flanking region, is found in all V. cholerae isolates examined. (b–d) The presence of genes VC0490, VC0512 and VC0514 encoded on VSP-II were investigated by PCR analysis. (e) The 3' flanking region of gene VC0517. Lanes: 1, 1 kb marker; 2–10, V. cholerae strains 569B, 0395, SM115, N16961, MO2, MO10, V46, V52, 468-83. NT, Non-toxigenic.

 


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Fig. 4. Southern hybridization analysis of V. cholerae VSP-II negative isolates. Lanes: 1–11, VSP-II-negative isolates confirmed by Southern hybridization by probing for VC0502; 12–13, VSP-II positive controls.

 
We found by PCR analysis with primer pair VC0489F/VC0489R that chromosomal DNA tested from all V. cholerae strains gave a 1·7 kb PCR band, indicating that this region is present in these isolates (Fig. 3). Thus, the VC0489 gene marks the 5' flanking region of VSP-II as shown by Dziejman et al. (2002). The gene directly downstream of VC0516 (integrase), VC0517, which encodes an RNA polymerase sigma factor, was assayed by PCR to determine whether it marked the 3' insertion site of the VSP-II region. Primer pair VC0517F/VC0517R was used to amplify a 1·8 kb PCR product from all strains tested (Fig. 3), indicating the presence of this region in all strains. Thus, VC0517 marks the 3' flanking region of VSP-II. By PCR analysis we have shown that VSP-II encompasses a 26·9 kb region, which is confined to V. cholerae El Tor and O139 serogroup isolates.

To investigate whether the region between VC0490 and VC0516 is empty or contains unique DNA in V. cholerae VSP-II-negative strains, we used primers VC0489F and VC0517R from core chromosome-specific DNA, lying respectively in the left and right chromosomal junction fragments of the 26·9 kb VSP-II. V. cholerae strains that lacked the 26·9 kb VSP-II region gave a 4 kb PCR product, while PCR analysis of V. cholerae VSP-II-positive strains did not produce a PCR product as expected since these strains contain the 26·9 kb region, which could not be amplified under the PCR conditions employed.

Bioinformatic analysis of the 26·9 kb VSP-II and VVI-I regions
The 26·9 kb region in V. cholerae El Tor and O139 isolates contained a total of 24 ORFs (Table 3). The overall GC content of VSP-I I (43 mol%) was much lower than the overall GC content of the V. cholerae genome (47 mol%) (Fig. 1). Each of the ORFs was analysed using the BLAST program (Altschul et al., 1997). Of the 24 ORFs identified, three ORFs (VC0490–VC0492) showed homology to three ORFs from Agrobacterium tumefaciens. Two ORFs (VC0513 and VC0514) showed homology to methyl-accepting chemotaxis proteins from the sequenced V. vulnificus strain YJ016. Of the 24 ORFs identified, 11 ORFs in this region were hypothetical proteins (VC0493–VC0510). Interestingly, VC0493–VC0498 and VC0504–VC0510 showed high sequence homology to a region in the sequenced V. vulnificus strain YJ016 (Chen et al., 2003) from VV0510–VV0516 and VV0518–VV0525, respectively. Eight ORFs in this region showed over 90 % amino acid sequence identity between the two species. In addition, V. cholerae ORF VC0516, which encodes an integrase, showed 93 % homology to VV0560 on the V. vulnificus genome. V. vulnificus ORF VV0560, similar to VC0516, was adjacent to a tRNA gene identified using Artemis. Next, we examined the region between VV0509 and VV0560 on the V. vulnificus genome in strain YJ016. In V. vulnificus strain YJ016, a clinical isolate from the blood of a patient with primary septicaemia isolated in Taiwan, 52 ORFs were identified (Fig. 5, Table 4). A second V. vulnificus genome sequence has recently become available, V. vulnificus strain CMCP6, a clinical isolate from South Korea (Chen et al., 2003; Kim et al., 2003). We examined the equivalent region, VV10636–VV10632, in strain CMCP6. Only two ORFs (VV10634 and VV10635) were present between VV10636 and VV10632 and these ORFs showed no homology to ORFs between VV0509 and VV0560 in strain YJ016 (Table 4, Fig. 5). Therefore, VV0509–VV0560 marked a 43·4 kb GEI in V. vulnificus, which we named VVI-I, that was only present in strain YJ016 and absent from strain CMCP6.


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Table 3. The 24 ORFs encompassing the 26·9 kb VSP-II island in V. cholerae N16961

 


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Fig. 5. (a) Regional variation in the mean proportion of GC content of VVI-I from V. vulnificus strain YJ016 based on a sliding window of 500 bp (Chen et al., 2003). (b–d) Schematic representation of the organization of VSP-II from V. vulnificus strain YJ016 (b), V. cholerae strain N16961 (c) and V. vulnificus strain CMCP6 (d). The position and direction of transcription of the ORFs are indicated by the direction of the arrows. The numbers refer to the genetic organization of the genes. Grey bars link homologous ORFs. Genes are pattern-coded according to their function.

 

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Table 4. The 52 ORFs encompassing the 43·4 kb VVI-I island in V. vulnificus YJ016

 
Among the 52 ORFs identified between VV0509 and VV0560 in V. vulnificus strain YJ016, were VV0510–VV0516 and VV0518–VV0525, which as stated previously showed homology of between 25 and 96 % at the amino acid level to V. cholerae VC0493–VC0498 and VC0504–VC0510, respectively (Tables 3 and 4). Within V. vulnificus ORFs VV0526–VV0560 there were a number of gene clusters; VV0528–VV0530 showed homology to sugar metabolism genes from Salmonella Typhimurium (STM3793–STM3791), VV0537–VV0540 showed homology to a phosphotransferase system from Yersinia pestis strain CO92 and ORFs VV0543, VV0554 and VV0555 showed homology to a sugar isomerase, sugar kinase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively (Table 4). Six transposase genes were also identified in this region, VV0531, VV0545, VV0546, VV0556, VV0558 and VV0559. Twenty-six ORFs were hypothetical or conserved hypothetical proteins. Similar to the 26·9 kb VSP-II region in V. cholerae El Tor and O139 serogroup isolates, the 43·4 kb VVI-I region in V. vulnificus strain YJ016 showed aberrant GC contents: 43 mol% as opposed to 46 mol% for the entire V. vulnificus genome (Fig. 5). So VVI-I is the first GEI to be identified in V. vulnificus strain YJ016.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our study has demonstrated that the 7·5 kb VSP-II region is part of a 26·9 kb island that encompasses ORFs VC0490–VC0516 (Table 3 and Fig. 1). In addition, we identified a 7·9 kb region within VSP-II that was homologous to a region on the V. vulnificus strain YJ016 genome. The 7·9 kb region of homology on the V. cholerae genome is located in the equivalent position on the V. vulnificus genome between a gene encoding a haemolysin (VC0489 and VV0508) and a gene encoding an RNA polymerase sigma factor (VC0517 and VV0561) (Fig. 5). In V. vulnificus strain YJ016 we examined the region between ORFs VV0508 and VV0561 and identified a 43·4 kb GEI which we named VVI-I. Zhang & Zhang (2004) examined V. vulnificus strain CMCP6 for anomalous sequence features and identified three regions that had GEI characteristics; none of the three GEIs corresponded to VVI-1. We found that VVI-I was absent in V. vulnificus strain CMCP6 where the site (VV10636–VV10632) is empty (Fig. 5).

In comparison to other known PAIs and GEIs, V. cholerae VSP-II and V. vulnificus VVI-I possessed most of the characteristics of laterally acquired genomic regions (Dobrindt et al., 2004; Hacker & Kaper, 2000). VSP-II and VVI-I have an atypical GC content of 43 mol% compared with 47 mol% for the entire V. cholerae genome and 46 mol% for V. vulnificus, they are inserted adjacent to a tRNA gene and each island contains a gene encoding a bacteriophage-like integrase (VC0516 and VV0560) (Fig. 5). The V. cholerae VSP-II region encodes transcriptional regulators, methyl-accepting chemotaxis proteins, a putative ribonuclease, a putative type IV pilin, a DNA repair protein, an integrase and a number of hypothetical proteins (Table 3, Fig. 1). The V. vulnificus VVI-I region encompasses genes homologous to VC0493–VC0510, with eight ORFs showing greater than 90 % amino acid identity (Table 3). This is significantly higher than the overall amino acid sequence similarity between the two species: approximately 80 % based on the housekeeping gene mdh. In addition VVI-I encodes a PTS mannose/fructose gene cluster, a number of sugar metabolism genes, an integrase and several transposase genes (Table 4). The role of these proteins in V. cholerae and V. vulnificus is unknown, but it is expected that GEIs contribute in some way to the fitness and survival of these species. The ecological niche of both V. cholerae and V. vulnificus is the aquatic ecosystem where conditions such as nutrient availability, temperature and pH are continuously changing. In this setting GEIs offer an opportunity for the organism to acquire a unique set of genes that may increase the chances of survival. For example, the acquisition of the ability to transport and metabolize additional sugars in the bacterial cell is a single-step gain of function event that could increase the fitness of a particular bacterium in a nutrient-limited environment.

VVI-I of V. vulnificus strain YJ016 appears to consist of three distinct regions. Region I is comprised of VV0510–VV0530 and is flanked by a transposase, VV0531. Region II from VV0532 to VV0544 is flanked by two transposase genes, VV0545 and VV0546. Region III from VV0547 to VV0557 is homologous to a sugar metabolism gene cluster on chromosome II of V. vulnificus strain CMCP6 (VV20914–VV20904), with amino acid identities of between 59 and 89 % (Table 4). This region also showed homology to a gene cluster in V. parahaemolyticus VP0073–VP0076 on chromosome 1 (Makino et al., 2003). Region III was flanked on each end by two IS elements, one adjacent to VV0560 (integrase) and the other flanking region II. Furthermore, if one examined the GC content across the VVI-I region, differences between the regions can be observed (Fig. 5). Thus, given the large number of transposase genes in VVI-I, it appears that different parts of the island may have been acquired at different times and from different sources. The extensive mosaic structure of PAIs has been revealed in a number of pathogenic bacteria (Ko et al., 2003; Welch et al., 2002; Dobrindt et al., 2002). It was suggested that VPI-2 (Jermyn & Boyd, 2002), which is unique to V. cholerae O1 serogroup isolates, is a mosaic, since the 5' region showed homology to a type 1 restriction modification system, the central region showed homology to amino sugar utilization genes and the 3' region had homology to numerous phage-like genes. Only the 3' region of the island was present in V. cholerae serogroup O139 isolates, indicating deletion of most of VPI-2 from these isolates (Jermyn & Boyd, 2002). Indeed, it has been shown recently that in Vibrio mimicus isolates, only ORFs VC1773–VC1784 of VPI-2 are present, suggesting that this region is highly unstable (Jermyn & Boyd, 2005).

To date three V. vulnificus biotypes have been described; biotype 1 is most frequently associated with pathogenicity in humans, whereas biotype 2 isolates cause disease in humans and eels, and biotype 3 has only been isolated in Israel from patients who handled St Peter's fish. Population genetic studies have identified a distinct eel-pathogenic clone; however, no clones showed any association with human infection (Gutacker et al., 2003). The significance of VVI-I to V. vulnificus virulence and survival needs to be examined, first by determining the occurrence of the island among isolates and examining the distribution pattern of the region among the various biotypes and genotypes.


   ACKNOWLEDGEMENTS
 
Research in E. F. B.'s laboratory is funded under the National Development Plan by the Higher Education Authority PRTLI-3 grant and Enterprise Ireland basic research grants. Research in J. M.'S and F. O'G.'s groups is supported by grants from the European Union (QLK3-2000-31759, QLK3-2001-00101 and QLK5-CT-2002-00914), the HEA PRTL1 programmes, Enterprise Ireland (SC/02/0520 and SC/02/517) and the Science Foundation of Ireland (02/IN.1/B1261).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 16 March 2004; revised 16 July 2004; accepted 5 October 2004.



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