Vibrio cholerae Pathogenicity Islands Form Circular Intermediates

The acquisition of novel DNA via horizontal gene transfer is known to play a significant role in both the emergence and recurrence of pathogenic bacteria.  A number of mobile genetic elements have been identified in isolates of pathogenic Vibrio chloreae, the causative agent of cholera, a disease which has resulted in thousands of deaths worldwide in the last decade alone.  Such mobile genetic elements include pathogenic genomic islands (PAIs), such as Vibrio pathogenicity island-1 (VPI-1, which encodes toxin-coregulated pili), VPI-2, Vibrio seventh pandemic island-I (VSP-I), and VSP-II.  These PAIs have been associated with endemic and pathogenic isolates of V. cholerae.  The nucleotide sequences of genomic islands display certain features which differentiate them from the rest of the bacterial genome.  Such features are used for identification purposes, and include the presence of mobility genes (e.g. integrases and transposases), a G+C (base) content which differs significantly from the overall content of the host, flanking direct repeat sites (that mark where incoming DNA recombined with the genome), association with a tRNA gene, and large regions of the chromosome that are present in one set of isolates, but absent in closely related isolates. 

Murphy & Boyd (2008) examined the genomic structures of VPI-2, VSP-I, and VSP-II, in the fully sequenced genomes of 12 different strains of V. cholerae.  Comparative analyses showed that the VPI-2 regions were highly conserved at the 5’ and 3’ insertion sites.  This prompted the authors to examine whether VPI-2 had the potential to excise from the genome (as previously shown in VPI-1), with subsequent formation of an extra-chromosomal, circular intermediate (CI).  The presence of both Phage-4-like integrase sequences (an enzyme that facilitates integration of transferred genetic material into host DNA), and two direct repeat sequences (one at the 5’ and one at the 3’ end, detected by comparative sequence analysis of VPI-2 positive and negative strains), suggested that an excision mechanism was present in the integrated VPI-2 region.  Therefore, the authors attempted to detect circular excision products of VPI-2 via inverse PCR and nested PCR (note: the primers used in both cases were designed to form products only if the VPI-2 region could excise and form a circular product).  No PCR product was detected in the initial, inverse PCR assays, but nested PCR (of inverse PCR products) resulted in amplified products from six strains, indicating that VPI-2 did indeed excise from its chromosomal insertion site, forming a circular excision product (although at low levels in the conditions applied).  The excision potential was likewise ascertained for the PAIs VSP-I and VSP-II, and the circular products were detected initially via inverse PCR, and subsequently confirmed via nested PCR.  All PCR products were sequenced to confirm the excision events.

The PAIs of several bacterial species have previously been shown to similarly excise from their chromosomal insertion sites.  Although the fate of circular excised islands is generally unknown, it has been shown that in some V. cholerae isolates, post-excision islands have the capability of reinserting into the genome at new locations.  Murphy & Boyd (2008) conclude that the excision and formation of CIs are probably the initial steps of PAI horizontal gene transfer (by transduction, transformation or conjugation), and go on to discuss the probable importance of chitin to the mechanisms underpinning naked DNA transfer in V. cholerae.  The authors also include analyses and investigations of specific PAI genes (the description of which is not possible in this short blog post).   In summary, Murphy & Boyd (2008) appear to have undertaken a thorough investigation which elucidates an important part of the process by which PAIs are transferred between V. cholerae strains.

Murphy R. A. & Boyd E. F. (2008) Three pathogenicity islands of Vibrio cholerae can excise from the chromosome and form circular intermediates. Journal of bacteriology 190: 636–47.