5 The role of quorum-sensing in the virulence of Pseudomonas ...

shows the success of the genomic island capture technique for the use in
characterisation of novel genomic islands. In conjugation with other data generated
within this project, the technique has been successfully used with bacterial species
other than P. aeruginosa.
3.5 Discussion
This project explored the characterisation of pathogenicity islands in vitro. The aim
of the project was to identify, capture and characterise genomic islands. To achieve
this aim involved development of the genomic island capture technique used by
Wolfgang et al. (2003) and the production of a generic system by targeting bacterial
species other than P. aeruginosa. To achieve these aims, the development of the
methodology was required to expand the use of the technique and to streamline the
recovery of the genomic islands.
3.5.1 Genomic island capture as a yeast-based technology
In terms of history of the genomic island capture technique, the current application
was piloted by Raymond et al. (2002a). Their first contribution to this application
was the introduction of a counterselectable marker, cycloheximide into the capture
vector (Raymond, Sims & Olson 2002b). They suggested (Raymond, Sims & Olson
2002b) and proved (Raymond et al. 2002a) that the use of a counterselectable
markers allowed capturing directly from genomic DNA by increasing the
percentage of clones that harboured a capture vector that had undergone
homologous recombination as opposed to those that harboured only the capture
vector. Raymond et al. (2002a) also piloted the first use of the technique to capture
variable genomic regions (O-antigen LPS region) from P. aeruginosa and were the
first to report using bacterial DNA in the capture process. Wolfgang et al. (2003)
adapted this technique further by modifying the structure of the capture vector.
Wolfgang et al. (2003) also used the method to target a variable region within the P.
aeruginosa genome and highlighted its use as a technique to capture genomic
islands. They constructed and used a capture vector to target the lys10 tRNA site
(p0975-0989capture) and captured genomic islands from a number of P. aeruginosa
strains. The technique as proposed by Wolfgang et al, 2003 is the basis of this
project.
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