Three pathogenicity islands of Vibrio cholerae can excise from the chromosome and form circular intermediates

Department of Microbiology, UCC, National University of Ireland, Cork, Ireland, Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA

^* To whom correspondence should be addressed. Email: fboyd{at}udel.edu.

	Abstract

The Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region integrated at a tRNA-serine locus and 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, a sialic acid metabolism region as well as neuraminidase (VC1784), which is a glycosylhydrolase known to release sialic acid from sialoglycoconjugantes 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 VPI-2 can form an extra chromosomal circular intermediate (CI) molecule after precise excision from its tRNA-serine attachment site. We constructed a knockout mutant of VC1758 (int) in 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 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 encodes 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.