The Vibrio Pathogenicity Island of Epidemic Vibrio cholerae Forms Precise Extrachromosomal Circular Excision Products

Abstract

The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. Like many pathogenicity islands, the VPI has at its termini a phage-like integrase gene (int), a transposase-like gene (vpiT), and phage-like attachment (att) sites, and is inserted at a tRNA-like locus (ssrA). We report that the VPI precisely excises from the chromosome and that its left and right ends join to form an extrachromosomal circular excision product (pVPI). Two-stage nested PCR analysis and DNA sequencing confirmed the int-attvpiT junction and that the core attP of pVPI is identical to the chromosomal VPI attR site. Excision was independent of toxR and toxT. Excision was independent of recA, suggesting that it is mediated by site-specific recombination. Interestingly, while excision was detected in int and vpiT mutants, excision was abolished in a double (int vpiT) mutant and was restored by plasmids containing genes for either recombinase. Excision results in deletion of A361 in the ssrA locus, which flanks the right junction of the VPI. Since A361 encodes U70 in the critical G ⅐ U base pair in the acceptor stem of the ssrA RNA that is the determinant for aminoacylation with alanine, this deletion might have deleterious effects on ssrA function. Also, vpiT may have undergone interchromosomal translocation or may represent an independent integration event, as it was found downstream of hutA in some isolates. Our results provide new insight into the molecular biology of the VPI, and we propose that the process of excision and circularization is important in the emergence, pathogenesis, and persistence of epidemic V. cholerae.Cholera, attributed to the bacterium Vibrio cholerae, is a diarrheal disease of humans that results in significant morbidity and mortality (25). Available records show that there have been seven worldwide pandemics of cholera (40). The current seventh pandemic began on the island of Sulawesi in Indonesia and then rapidly spread throughout Asia (1, 24). Because of its high death-to-case ratio, transmissibility, and persistence in the environment and its ability to occur in explosive epidemic form, V. cholerae continues to be a public health concern.Only toxigenic strains of V. cholerae that possess two essential virulence gene clusters, i.e., the CTX element, which is a prophage encoding cholera toxin (39, 49), and the Vibrio pathogenicity island (VPI) (26, 30), can cause epidemic cholera. The chromosomal VPI is 41.2 kb in size in both sixthpandemic (classical biotype) and seventh-pandemic (El Tor biotype) strains and encodes 29 potential proteins (26, 27). The VPI encodes proteins with essential roles in virulence, such as toxin-coregulated pili (TCP) (17, 44), and proteins that regulate virulence, such as ToxT, TcpP, and TcpH (4,6,7,14). In addition, the VPI contains several open reading frames with no known or demonstrated function.The VPI has many features that are typical of pathogenicity islands (PAIs) (2, 12, 13). It has a low percent GϩC content (35%) compared to the re...

Characterization of pathogenicity island prophage in clinical and environmental strains of Vibrio cholerae

“…The implication from these results may be that: (i) some environmental strains could have acquired VPI phage; however, due to phages' size limitation they did not acquire the whole phage genomics; and (ii) the ability of V. cholerae to acquire, integrate and excise the VPI cluster is mediated by the VPI-encoded recombinase int and vpiT (Karaolis et al, 1998;Rajanna et al, 2003). Generation of amplicons with right and left junction primers suggests that VPIs have been integrated in the large chromosome of these isolates at sites identical to those of O1 strains (Fasano et al, 1991).…”

VEJφ, a novel filamentous phage of Vibrio cholerae able to transduce the cholera toxin genes

“…The receptor of this phage is the toxin co-regulated pilus (TCP), a type IV fimbria essential for colonization of the human intestine by V. cholerae (Waldor & Mekalanos, 1996). The genes required for TCP expression and assembly at the bacterial surface are located in the vibrio pathogenicity island (VPI), which has also been horizontally acquired by this pathogen during evolution, although its mechanism of transmission is still controversial (Faruque et al, 2003;Karaolis et al, 1998;Karaolis & Kaper, 1999;Lee, 1999;Rajanna et al, 2003).…”

CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules

“…Both VPI-1 and VPI-2 were shown to excise as complete units from the bacterial chromosome (10)(11)(12)(13). The mechanisms of excision for both islands have recently been determined (13).…”