Genomic mosaicism in the opportunistic pathogen pseudomonas aeruginosa and its contribution to virulence

Pseudomonas aeruginosa is a ubiquitous Gram negative opportunistic pathogen which belongs to the gamma subclass of proteobacteria. P. aeruginosa genomes consist of a highly conserved core component making up ~90% of the total genome and a highly variable accessory genome that makes up the remaining ~10% of the organism's DNA blueprint.;The extent and nature of tRNA-integrated genomic islands in the available sequenced P. aeruginosa genomes and clinical isolates were defined by both in silico comparative genomics methods and in vitro approaches. These investigations demonstrated that certain tRNA sites seemed to be favoured for integration and that most strains exhibited divergent tRNA site island-occupation profiles.;The contribution of three P. aeruginosa genomic islands PAPI-1, PAPI-2 and the tRNAPro21 island in strain PA14 to fitness and virulence were evaluated. By creating 'en bloc' deletant mutants that lacked islands and testing the resulting phenotype of the mutants alongside the wild-type parent, three islands (PAPI-1, PAPI-2 and tRNAPro21 island) were demonstrated to be important to full virulence of strain PA14.;The dynamics of site-specific integration of the PAPI-1 island into both of its cognate target attB sites were examined. A quantitative real-time PCR approach was used to measure the copy number of all 'states' of PAPI-1: chromosomally-integrated forms present at each of the two tRNALys sites, extra-chromosomal circular entities, and empty versions of the two attB sites. The pattern of occupation was clearly dynamic. Prior occupation of the tRNA Lys10 site by an alternative genomic island in PAOl and PA14 appeared to hinder integration of PAPI-1 into this occupied tRNA Lys10 site through an as yet unknown mechanism. This hypothesis was supported by evidence that the deletion of the original occupying island allowed PAPI-1 to integrate at high frequency into this site. However, surprisingly this effect was found to be variable as several independently derived deletant mutants behaved distinctly. These studies also identified a potential link between the number of chromosomally-integrated copies of PAPI-1 and the stability of the island.;Several approaches, including the introduction of a third attB site within a 'neutral' genomic location combined with DNA signature copy number assays and pulsed field gel electrophoresis to identify large scale inversions, deletions or translocations involving rrn operons or tRNA genes were used to further investigate this mysterious 'dynamic-yet-stable genome'. The results demonstrated that rrn operon-mediated inversions are a constantly occurring in P. aeruginosa populations. Each strain does not have a single genomic orientation but rather a dominant orientation with other genomic variants also present within the population.