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

3.2 Capture experiments involving clinical bacterial strains
After standardising the technique, putative genomic islands within bacterial strains
with un-sequenced genomes (clinical isolates) were targeted. This section proceeds
by describing how the suspected genomic islands were identified. This is followed
by a summary of the genomic islands targeted within the clinical isolates and brief
descriptions of three targets. Two targets are excluded from this section (E106-serW
and E105-leuX), as they are discussed and analysed within their own section.
The clinical isolate genomic island targets were determined by tRIP PCR (tRNA
site interrogation for pathogenicity islands, prophages and other genomic islands)
and SGSP PCR (single genome specific primer). The tRIP PCR and SGSP data
were gathered by Ali Thani (Thani A, PhD thesis, University of Leicester) and
Ewan Harrison (Harrison E, PhD thesis, University of Leicester, unpublished) for E.
coli and P. aeruginosa respectively. The tRIP PCR technique is particularly usually
for screening a large number of bacterial strains for genomic islands. The technique
involves using a set of primers, one upstream and the other downstream of the
tRNA site within the conserved flanks. If the site is empty or contains an islet of
≤5kb, it will lead to a successful amplification of DNA, if there is an insert present
then the PCR will fail. The PCR negative strains are then analysed further by SGSP
PCR to get a snapshot of the structure of the genomic island and then analysed to
determine whether there are similar islands already present within sequenced
strains. The clinical isolate targets and the capture vectors used are listed in Table
3-4. The captured insert size is estimated from restriction digestion with I-SceI
resolved on a 0.6% agarose gel.
Number Organism Capture Vector Size of insert
1 E. coli E105 leuX ~11kb
2 E. coli E106 serW ~14kb
3 E. coli E105 serT Unsuccessful
4 P. aeruginosa KR115 lys10 ~9kb
5 P. aeruginosa KR159 lys10 ~9kb
Table 3-4 shows clinical isolate strains targeted for genomic island capture. The sizes of the
captured inserts were estimated from I-SceI restriction digest resolved on a 0.6% agarose gel.
58