Bioinformatics Advance Access originally published online on January 27, 2006
Bioinformatics 2006 22(8):905-910; doi:10.1093/bioinformatics/btl015
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Genomic island identification in Vibrio vulnificus reveals significant genome plasticity in this human pathogen
Department of Microbiology, University College Cork, National University of Ireland Cork, Ireland
*To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Genomic islands (GIs) are large chromosomal regions present in a subset of bacterial strains that increase the fitness of the organism under specific conditions. We compared the complete genome sequences of two Vibrio vulnificus strains YJ016 and CMCP6 and identified 14 regions (ranging in size from 14 to 117 kb), which had the characteristics of GIs. Bioinformatic analysis of these 14 GI regions identified the presence of phage-like integrase genes, aberrant GC content and genome signature (dinucleotide frequency) within each GI compared with the core genome indicating that these regions were acquired from an anomalous source. We examined the distribution of the nine GIs from strain YJ016 among 27 V.vulnificus isolates and found that most GIs were absent from the majority of these isolates. The chromosomal insertion sites of three GIs were adjacent to tRNA sites, which contained novel horizontally acquired DNA in all six available sequenced Vibrionaceae genomes.
Contact: f.boyd{at}ucc.ie
Supplementary information: Supplementary data are available at Bioinformatics online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The area of comparative genomic analysis has developed exponentially in recent years, and this has been largely facilitated by the advent of genome sequencing. To date there are over 285 bacterial genomes, fully annotated, and available in public databases. In particular, the annotated genome sequence is available for five species of the bacterial family Vibrionaceae, including the complete sequence of two strains of Vibrio vulnificus strains YJ016 and CMCP6. V.vulnificus is a human pathogen that is highly invasive, causing fulminant pulmonary septicemia, with mortality rates as high as 75%, one of the highest death rates of any foodborne disease (Gulig et al., 2005; Linkous and Oliver, 1999). V.vulnificus infection is most lethal in individuals who have a preexisting chronic illness, are immunocompromised or have preexisting liver disease (Gulig et al., 2005; Haq and Dayal, 2005; Linkous and Oliver, 1999). The complete genome sequence of V.vulnificus strain YJ016, a clinical biotype 1 isolate from Taiwan, was published in 2003 (Chen et al., 2003). A second V.vulnificus genome sequence is also available in the database, strain CMCP6 a biotype 1 clinical isolate from South Korea (Kim et al., 2003). In addition, the genome sequence of Vibrio parahaemolyticus RIMD2210633, Vibrio cholerae N16961, Vibrio fischeri ES114 and Photobacterium profundum SS9 have all been published (Heidelberg et al., 2000; Makino et al., 2003; Ruby et al., 2005; Vezzi et al., 2005).
Genomic islands (GIs) are a group of large chromosomal regions that are acquired by horizontal gene transfer and are found to be compositionally biased from their host genome in terms of GC content, genome signature (dinucleotide frequency) and codon usage patterns (Dobrindt et al., 2004). GIs encode genes that may increase fitness of the bacterium in a particular environment, e.g. virulence genes present on pathogenicity islands. GIs are usually present in a subset of a bacterial population and absent from closely related species or strains of the same species. As well as aberrant DNA composition, GIs have the general characteristics of encoding a bacteriophage-like integrase, being flanked by repeat sequences and inserting adjacent to tRNA genes probably indicating a similar mechanism of chromosomal integration (Dobrindt et al., 2004).
In this study, we compared the complete genome sequence of V.vulnificus strain YJ016 with V.vulnificus strain CMCP6 using Artemis comparison tool (ACT) and WebACT to identify GIs unique to YJ016. We aimed to determine the extent of genomic plasticity that exists in this species and the level of horizontal gene transfer. Furthermore, we analysed a collection of 27 V.vulnificus clinical and environmental isolates to assess the distribution and predominance of these GIs, enabling us to determine the possible significance and the role they may play in the success of the species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Complete nucleotide sequences and annotations for the Vibrionaceae were retrieved and downloaded from NCBI ftp site at (http://ftp.ncbi.nlm.nih.gov). Initial intra-species whole genome comparisons of V.vulnificus YJ016 with V.vulnificus CMCP6 were generated using WebACT and identified a large number of chromosomal rearrangements, mainly inversions across the replication axis, which is highly unusual for intra-species comparisons (Abbott et al., 2005). Strand composition bias [(G – C)/(G + C)] was analysed for each V.vulnificus genome through ACT (Fig. 1). BLAST analysis of Escherichia coli oriC was used to identify the precise location and orientation of the oriC in the Vibrio genomes. The local alignment tool ClustalW (http://www.ebi.ac.uk/clustalW) was used to align sequences around the origin of each Vibrio species. This enabled us to reorder the CMCP6 genome sequence starting with the origin and with the positive Crick strand determined by the orientation of the ori motif sequences. With the reordered CMCP6 genome, whole genome comparative analysis was performed using Artemis Comparison Tool v3 from the Sanger Institute http://www/sanger.ac.us/Software/ACT/ (Fig. 1) (Carver et al., 2005; Rutherford et al., 2000). Gene pairs were generated for ACT by tBLASTx and BLASTn analysis for both V.vulnificus genomes (Altschul et al., 1997). Regions unique to V.vulnificus strain YJ016 were further analysed for sequence similarities using the BLAST algorithm.
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Several criteria were used in this study to determine the presence of GIs in what amounted to an integrated genomic approach. These consisted of the traditional selection criteria of aberrant G + C percentage and codon usage, presence of insertion elements, proximity of tRNA and the size of the unique region. Putative regions that fulfilled these criteria were then subjected to compositional bias of dinucleotide frequency analysis using the web-based application deltarho (http://deltarho.amc.uva.nl) (van Passel et al., 2005a,b). Deltarho calculates the genomic dissimilarity values
* (the average dinucleotide relative abundance difference) between input sequences (GIs) and the V.vulnificus YJ016 and CMCP6 genome sequence (van Passel et al., 2005a,b), which is based on the work of Karlin (Karlin and Ladunga, 1994; Karlin, 2001). A high genomic dissimilarity (*) between an input sequence (genomic island) and the host genome sequence indicates a heterologous origin of the input sequence. Since the length of the input sequence is important in calculating the relevance of the value of *, van Passel's method also calculates the plot position (in percentage) of the input sequence in the * versus fragment number plot of the complete genome divided in non-overlapping fragments of equal size as the input sequence (van Passel et al., 2005a,b). This plot position depicts the percentage of the genomic fragments that have a lower genomic dissimilarity than the input sequence. In this way, the high * values of various GIs can be shown (van Passel et al., 2005a,b). Total genomic DNA from each of the 27 V.vulnificus isolates was extracted using the G-nome DNA isolation kit (Bio 101 Systems). PCR assays to determine the presence of 9 GIs among the 27 V.vulnificus natural isolates were carried out using 40 primer pairs designed from the genome sequence of V.vulnificus strain YJ016 (Supplementary data Table 2). PCR assays were performed in volumes of 25 µl containing 10 ng genomic DNA, 10 pmol primer and 1 U Taq DNA polymerase. The amplification conditions were pre-incubation at 96°C for 1 min, followed by 30 cycles of 94°C for 30 s, 48–55°C (depending on the primer pair) for 30 s and 72°C for a time chosen based on the size of the expected fragment (1 min/kb). All PCRs were amplified in a PTC-200 Peltier Thermal Cycler (MJ Research). Southern hybridization was carried out with DNA probes, generated by PCR from the reference strain YJ016, on GI-negative strains to confirm PCR negative results. Briefly, DNA from each strain was digested with the restriction enzyme EcoR I (Roche Molecular Biochemicals) and the fragments were separated by electrophoresis in 0.6% TBE agarose. DNA fragments were transferred to nylon membrane under positive pressure (Stratagene Posiblotter, La Jolla, CA). Probe DNAs were labeled using the ECL direct nucleic acid labeling system (Amersham Pharmacia Biotech) and positive hybridization was detected by the ECL chemiluminescent substrate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Identification of chromosomal regions unique to V.vulnificus YJ016
In order to identify GIs that may be unique to this important human pathogen and that may mark subsets of isolates, we compared the whole genome sequence of V.vulnificus YJ016 with the genome sequence from V.vulnificus strain CMCP6 using ACT (Fig. 1). ACT analysis of V.vulnificus YJ016 with CMCP6 identified 10 regions of >10 kb that were present in YJ016 but absent from CMCP6; a superintergon (SI) and nine additional regions which we named V.vulnificus island-I (VVI-I) to VVI–IX (Fig. 1, Table 1). The nine regions identified ranged in size from 14 to 117 kb and were dispersed throughout the genome, with the majority (seven) found on chromosome 1. Of the nine VVI regions uncovered in this study, only VVI-I was previously identified (O'Shea et al., 2004a,b). Three GIs VVI-I, VVI-V and VVI-VII inserted adjacent to a tRNA-met, a tmRNA (ssrA) and a tRNA-ser locus, respectively (Table 1). All GIs, except VVI-IV, contained integrase or transposase genes, which may indicate possible mechanisms of insertion (Table 1). Comparative genome analysis of V.vulnificus CMCP6 with YJ016 identified seven regions unique to CMCP6 that were absent from YJ016, a SI, a prophage and five regions that showed the characteristics of GIs, which we named VVI-1 to VVI-5 (Table 1). Three GIs from CMCP6, VVI-1, -2, -3 inserted adjacent to a tmRNA, a tRNA-arg and a tRNA-met loci, respectively (Table 1).
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Compositional bias of regions unique to V.vulnificus YJ016
Using the program developed by van Passel et al., (2005a,b), we examined the compositional dissimilarities among the 14 GIs identified by ACT analysis compared with the host genome (Table 1). Five GI regions from YJ016 had a GC content ranging from 39 to 43%, which was much lower than the overall chromosomal 46% GC (Table 1, Supplementary data Fig. 1). Of the five CMCP6 GIs identified, four GIs had a GC content lower than that of the core genome ranging from 38 to 44%. Among the nine GIs identified in strain YJ016, six GIs gave a high genomic dissimilarity value (
*) compared with the average for chromosome 1 of V.vulnificus YJ016 (which was 38) (Table 1, Supplementary data Fig. 1). Of the five CMCP6 GIs identified only one region (VVI-3) gave a low genomic dissimilarity value compared with the average for chromosome 1 of CMCP6 (which was 34) (Table 1, Supplementary data Fig 1). Analysis of the percent genomic fragments with lower genomic dissimilarity (*) than those of the 14 GIs identified only one region (VVI-VI) that was similar to the core genome (Table 1).
Comparative phylogenetic analysis
Each ORF present on the nine GIs identified in YJ016 was analysed systematically by BLAST analysis to determine whether these regions were present in sequenced members of the family Vibrionaceae; V.cholerae N16961, V.parahaemolyticus RIMD2210633, V.fischeri ES114 and P.profundum SS9. For the majority of GIs, no homologues were present in these sequenced genomes examined. Several ORFs with similarity to VVI-I and VVI-II were identified in V.cholerae N16961. The homologous insertion sites for five regions, VVI-I, VVI-II, VVI-III, VVI-VI and VVI-VIII, were empty in V.vulnificus CMCP6. For example, the 37 kb VVI-II region is absent from the other Vibrionaceae sequenced genomes and the homologous core flanking genes were adjacent to each other indicating the absence of novel DNA (Fig. 2A). In contrast, the homologous insertion sites of VVI-I, VVI-V and VVI-VII, the tRNA-met, tmRNA and tRNA-ser loci respectively, all contained novel DNA amongst the five Vibrionaceae genomes examined. For example, VVI-V from strain YJ016 inserted adjacent to a tmRNA, and this chromosomal insertion site contained novel DNA in V.vulnificus CMCP6 and the other four Vibrio species (Fig. 2B). Similarly, at the homologous tRNA-met and tRNA-ser loci in V.parahaemolyticus RIMD2210633, V.cholerae N16961 and V.fischeri ES114 novel DNA was present (data not shown).
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Distribution of GIs among natural isolates of V.vulnificus
To determine whether the nine GIs are unique to strain YJ016 or if they are prevalent among clinical or environmental V.vulnificus isolates, we examined their distribution among 27 isolates. We preformed PCR assays using a range of primer pairs and DNA probes encompassing the GIs and their insertion site flanking core chromosomal genes (Supplementary data). Our analyses showed that six of the GIs (VVI-I, -III, -IV, -V, -VII, -IX) were unique to YJ016 since we failed to detect these regions in any of the 27 V.vulnificus isolates examined. Three GIs were present in a very limited number of V.vulnificus isolates (average 2 strains). Primer pairs consisting of a forward primer designed from the 5' core chromosomal flanking gene and a reverse primer designed from the 3' flanking gene for each of the GIs was used to examine the GI insertion sites in GI-negative strains. PCR products were obtained with DNA templates from some GI-negative strains indicating an empty insertion sites. However, for many GI-negative strains tested, no PCR product was obtained suggesting that the insertion sites in these strains may contain novel DNA that could not be amplified under conventional PCR conditions used in this study.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A key problem in the monitoring and identification of pathogenic V.vulnificus isolates is the availability of molecular techniques such as DNA probes and PCR assays for the differentiation of pathogenic and non-pathogenic isolates. The identification of genomic regions unique to V.vulnificus clinical or environmental isolates may be key to addressing this problem and help our understanding of how pathogenic strains originate and evolve. In this study, we identified nine chromosomal regions that were present in V.vulnificus YJ016 but absent from V.vulnificus CMCP6. At three tRNA loci, tRNA-met, tmRNA and tRNA-ser, novel DNA was present in strain YJ016. The tRNA-met site in CMCP6 contains no novel DNA, however at the tmRNA locus, a novel 37 kb genomic region was present, and at the homologous tRNA-ser site, a 17 kb region was present. At all three tRNA loci either a pathogenicity or genomic island was present in V.cholerae N16961 and V.parahaemolyticus RIMD2210633. For example, in V.cholerae the seventh pandemic island (VSP)-II, the Vibrio pathogenicity island-1 and the VPI-2 are present at the tRNA-met, tmRNA and tRNA-ser loci respectively. These three pathogenicity islands are all present predominantly in toxigenic V.cholerae isolates associated with epidemic cholera (Dziejman et al., 2002; Karaolis et al., 1998; O'Shea et al., 2004a,b; Jermyn and Boyd, 2002, 2005). At the same loci in V.parahaemolyticus three novel GIs are present, which are only associated with post-1995 pandemic isolates (Hurley and Boyd, unpublished data). It has long been appreciated that tRNA loci serve as integration sites for a range of genetic elements, particularly those encoding integrases of the tyrosine recombinase family (Campbell, 1992; Hacker et al., 1997; Hou, 1999; Williams, 2002). However, it has only recently been demonstrated that there is an appreciative bias in specificity for the tmRNA locus than for any other type of tRNA gene (Williams, 2003). Indeed in our study, in all six genomes of the family Vibrionaceae examined, novel DNA was present at this site.
The absence of six of the GIs and the presence of three GIs in only a small fraction of the 27 isolates examined suggests two possible scenarios; first, that the strain YJ016 is unusual in containing so much novel DNA or, second, that all strains of V.vulnificus contain a large amount of strain specific DNA. We examined the integration sites of the nine GIs in V.vulnificus GI-negative isolates by PCR amplification using primer pairs designed from GI flanking genes. For many strains no PCR products were obtained indicating the possible presence of a large fragment of DNA that was outside the limits of our PCR conditions. These data suggest that the V.vulnificus genome has a highly flexible gene pool which needs to be further investigated. We were surprised to find that none of the GI identified in this study marked clinical, environmental or biotype specific strains indicating that in our study pathogenic strain could not be differentiated from non-pathogenic strains. Since others have shown that differentiation of clinical and environmental isolates may be possible, it may be necessary to extent our analysis to a larger set of non-clinical environmental samples (Nilsson et al., 2003; Rosche et al., 2005). Our results are in contrast to studies in V.cholerae and V.parahaemolyticus, where it is known that regions encoding virulence factors are only associated with a subset of isolates. The presence of so many unique regions in both YJ016 and CMCP6 and the possibility that other V.vulnificus strains may contain additional regions leads to questions regarding GI function and mechanisms of acquisition. Many of the GIs in our study appear to be involved in sugar transport and metabolism (putative Metabolic islands); others contain multidrug resistance genes (putative Resistance islands) and two GI encoded possible virulence factors (putative Pathogenicity islands), which indicates the diversity of roles that these regions may play in V.vulnificus survival. A definitive mode of GI transfer and uptake has not been described but possible mechanisms include transduction and transformation in the natural environment. The aquatic environment is teeming with viral particles and free DNA, which gives ample opportunity for the acquisition of novel DNA. The recent report that chitin, an abundant biopolymer in the aquatic environment, induces natural competence in V.cholerae may help to example the high level of genomic diversity (Meibom et al., 2005). V.vulnificus has a complex life-style involving a free-living state, an animal or particle association state as well as a viable but non-culturable state. The finding of large chromosomal regions unique to strains of V.vulnificus YJ016 and CMCP6 suggests a role in evolution possibly increasing strain diversity and adaptability of the organisms to new and changing environments.
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Acknowledgments |
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This work was supported by a Science Foundation Ireland (SFI) Research Frontiers Programme grant 05/RFP/Gen0006 and an SFI Investigator Programme grant 04/IN3/B651. F.J.R. is funded by an EMBARK IRSCET Postdoctoral fellowship.
Conflict of Interest: none declared.
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FOOTNOTES |
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Associate Editor: Thomas Lengauer
Received on October 14, 2005; revised on December 19, 2005; accepted on January 20, 2006
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