Three Pathogenicity Islands of Vibrio cholerae Can Excise from the Chromosome and Form Circular Intermediates

Abstract

Vibrio pathogenicity island-2 (VPI-2) is a 57-kb region integrated at a transfer RNA (tRNA)-serine locus that encompasses VC1758 to VC1809 on the V. cholerae N16961 genome and is present in pandemic isolates. VPI-2 encodes a P4-like integrase, a restriction modification system, a Mu phage-like region, and a sialic acid metabolism region, as well as neuraminidase (VC1784), which is a glycosylhydrolase known to release sialic acid from sialoglycoconjugates to unmask GM1 gangliosides, the receptor for cholera toxin. We examined the tRNA-serine locus among the sequenced V. cholerae genomes and identified five variant VPI-2 regions, four of which retained the sialometabolism region. Three variant VPI-2 regions contained a type three secretion system. By using an inverse nested PCR approach, we found that the VPI-2 region can form an extrachromosomal circular intermediate (CI) molecule after precise excision from its tRNA-serine attachment site. We constructed a knockout mutant of VC1758 (int) with V. cholerae strain N16961 and found that no excision PCR product was produced, indicating that a functional cognate, VPI-2 integrase, is required for excision. The Vibrio seventh pandemic island-I (VSP-I) and VSP-II regions are present in V. cholerae O1 El Tor and O139 serogroup isolates. Novel regions are present at the VSP-I insertion site in strain MZO-3 and at the VSP-II insertion site in strain 623-39. VSP-II is a 27-kb region that integrates at a tRNA-methionine locus, is flanked by direct repeats, and encodes a P4-like integrase. We show that VSP-II can excise and form a CI and that the cognate VSP-II integrase is required for excision. Interestingly, VSP-I is not inserted at a tRNA locus and does encode a XerDC-like recombinase, but similar to VPI-2 and VSP-II, VSP-I does excise from the genome to form a CI. These results show that all three pathogenicity islands can excise from the chromosome, which is likely a first step in their horizontal transfer.

Deep-sea hydrothermal vent bacteria related to human pathogenic Vibrio species

“…The 12-kb insert did not show significant match with any sequences in the National Center for Biotechnology Information GenBank database, except for a 103-bp region with 78% sequence similarity to a transcriptional regulator of V. parahaemolyticus. Recent analyses of the VC1773 to VC1810 region of this island suggest this is a hot-spot for novel DNA insertion (58). The presence of virulence factors, including two reported in V. cholerae, namely pre-CTXΦ and VPI-2 in the noncholera Vibrio antiquarius from a deep-sea environment, suggests their multifaceted role outside the human host; that is, ecological function in the natural habitat and alternate evolutionary origins apart from their core genome.…”

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

“…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). Murphy & Boyd (2008) showed that all three pathogenicity islands can excise from the chromosome, which is likely a first step in their horizontal transfer. Boyd et al (2000) suggest that Vibrio mimicus may be a significant reservoir for VPI phage and may play an important role in the emergence of new toxigenic V. cholerae isolates.…”

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

“…The major virulence factors of V. cholerae, the aetiological agent of Asiatic cholera, are encoded by genes within A+T-rich horizontally transmissible genetic elements (Davis & Waldor, 2003;McLeod et al, 2005;Murphy & Boyd, 2008). These genes are regulated by several environmental signals to ensure that their products are expressed when the bacterium arrives at appropriate sites in the host and that they are repressed elsewhere (Lee et al, 1999;Schild et al, 2007).…”