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Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.The metabolically versatile Pseudomonas aeruginosa is an opportunistic pathogen of plants, animals, and humans and is ubiquitously distributed in soil and aquatic habitats. The common reference strain is P. aeruginosa PAO1, a spontaneous chloramphenicol-resistant mutant of the original PAO strain (earlier called "P. aeruginosa strain 1") that had been isolated in 1954 from a wound in Melbourne, Australia (9, 10). This PAO1 strain from Bruce Holloway's laboratory has become the reference strain for Pseudomonas genetics and functional analyses of the physiology and metabolism of this gammaproteobacterium. A genetic map of its chromosome was generated by exploiting the mechanisms of gene exchange in bacteria, i.e., transduction and conjugation (11). With the advent of pulsedfield gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). By 2000 the PAO1 strain had been completely sequenced (36). Thereafter, the genome annotation has been continually updated and the database content and functionality have been expanded to facilitate accelerated discovery of P. aeruginosa drug targets and vaccine candidates (38). Two near-saturation libraries of transposon insertion mutants have been constructed in P. aeruginosa PAO1 as a global resource for the scient...

The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNA Lys genes, pKLC102 is incorporated into the tRNA Lys gene only close to the pilA locus. Targeting of the other tRNA Lys copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNA Gly -associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.

Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNAGly genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.Genetic variability within bacterial species can be the result of nucleotide substitutions, intragenomic reshuffling, and acquisition of DNA sequences from another organism (3). The considerable impact of the last strategy, termed horizontal gene transfer, on microbial evolution and its integral role in the diversification and speciation of the bacteria has become apparent from recent analyses based on the growing pool of genomic sequence information (7,18,23,28). Prominent examples are the pathogenicity islands of many obligatory pathogens (14). These chromosomally encoded regions typically contain large clusters of virulence genes not present in closely related nonpathogenic strains and can, upon integration, transform a benign organism into a pathogen. Whereas the molecular mechanism of chromosomal integration has been resolved for some conjugative transposons and bacteriophages and details about the transmissibility of conjugative plasmids are well known, the evolution and mobility of gene islands remain obscure (14). Often these DNA blocks are integrated adjacent to or within tRNA genes, and some contain a phage-related integrase gene near one end, suggesting that gene islands may have been generated by a phage or by a plasmid with integrative functions (14, 42). Nevertheless, the comparative sequence analysis of gene islands so far have not pointed to any common genetic repertoire that confers transmission and acquisition.The gram-negative bacterium Pseudomonas aeruginosa is ubiquitously distributed in aquatic and soil habitats, and it is an opportunistic pathogen for plants, animals, and humans (38). No correlation between certain P. aeruginosa clones and disease habitats or environm...

SummaryPseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS 6100 -induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS 6100 into wbpM , pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.

The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 89 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB-attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3' end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNA Lys genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome ; the att site of the phnAB-oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone-and strainspecific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.

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