Department of Microbiology, UCC, National University of Ireland, Cork, Ireland
Correspondence
E. Fidelma Boyd
f.boyd{at}ucc.ie
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ABSTRACT |
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INTRODUCTION |
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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 (VC0175VC0185) 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 (VC0490VC0497), 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 (VCO490VC0497) encompasses a 26·9 kb region (VC0490VC0516) in V. cholerae biotype El Tor and O139 serogroup isolates.
The 5' region of V. cholerae VSP-II, ORFs VC0493VC0498 and VC0504VC0510, shows homology to a region in V. vulnificus strain YJ016, ORFs VV0510VV0516 and VV0518VV0525, respectively. We determine that in V. vulnificus strain YJ016, ORFs VV0510VV0525 are part of a 43·4 kb genomic island (GEI) (VV0509VV0560) named Vibrio vulnificus island-I (VVI-I) which encompasses a number of transport and sugar metabolism genes.
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METHODS |
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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 (49·658 °C) and 14 min of primer extension at 72 °C.
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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).
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RESULTS |
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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 (VC0490VC0492) 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 (VC0493VC0510). Interestingly, VC0493VC0498 and VC0504VC0510 showed high sequence homology to a region in the sequenced V. vulnificus strain YJ016 (Chen et al., 2003
) from VV0510VV0516 and VV0518VV0525, 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, VV10636VV10632, 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, VV0509VV0560 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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DISCUSSION |
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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 VC0493VC0510, 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 VV0510VV0530 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 (VV20914VV20904), with amino acid identities of between 59 and 89 % (Table 4). This region also showed homology to a gene cluster in V. parahaemolyticus VP0073VP0076 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 VC1773VC1784 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.
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ACKNOWLEDGEMENTS |
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Received 16 March 2004;
revised 16 July 2004;
accepted 5 October 2004.
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