Chromosomal rearrangement in PAO1 repositions the replication terminus relative to the origin. (a) Schematic of PAO1 and PA14 chromosomes. The region with the same orientation in both strains is shown with a thick red line; a thin blue line represents the inverted region. Arrows represent the positions and orientations of the four ribosomal RNA clusters. PCR products designed with unique sequences flanking each rRNA cluster are indicated by numbers next to each arrow. PCR products 1 and 2 (purple numbers) are diagnostic for the PAO1 chromosome structure; PCR products 3 and 4 (black numbers) are diagnostic for the PA14 chromosome structure. The position of the presumptive terminus of replication in each strain is indicated by an orange triangle marked with the corresponding position along the chromosome (expressed as the percentage of the whole chromosome, starting from the origin of replication and moving in a clockwise direction). (b) Diagnostic long-range PCR spanning each ribosomal RNA repeat demonstrates the inversion in PAO1. PCR products corresponding to the numbers indicated in panel a were generated using genomic DNA from PAO1 (left panel) or PA14 (right panel). PCR products 1 and 2 were obtained only when using PAO1 genomic DNA (the weak background band for product 1 seen using PA14 as a template was also observed for 18 additional P. aeruginosa strains tested). PCR products 3 and 4 were obtained only when using PA14 genomic DNA. PCR products 5 and 6 were obtained from both strains. (c) GC skew analysis was performed using 1 kb windows, and the cumulative GC skew is shown on the y-axis as a function of chromosomal location (x-axis) for PA14 (black line) and PAO1 (purple line). The position of the peak indicates the likely position of the terminus of replication. For PA14 the peak is centered in the middle of the chromosome (at 49.2% of the genome, between coordinates 3,219,001 and 3,220,000), whereas the peak for PAO1 is offset with respect to the origin of replication as a result of the inversion (occurring at 38.8% of the genome, between coordinates 2,428,001 and 2,429,000). PCR, polymerase chain reaction.