Pseudomonas aeruginosa Genomic Structure and Diversity

Abstract

The Pseudomonas aeruginosa genome (G + C content 65-67%, size 5.5-7 Mbp) is made up of a single circular chromosome and a variable number of plasmids. Sequencing of complete genomes or blocks of the accessory genome has revealed that the genome encodes a large repertoire of transporters, transcriptional regulators, and two-component regulatory systems which reflects its metabolic diversity to utilize a broad range of nutrients. The conserved core component of the genome is largely collinear among P. aeruginosa strains and exhibits an interclonal sequence diversity of 0.5-0.7%. Only a few loci of the core genome are subject to diversifying selection. Genome diversity is mainly caused by accessory DNA elements located in 79 regions of genome plasticity that are scattered around the genome and show an anomalous usage of mono- to tetradecanucleotides. Genomic islands of the pKLC102/PAGI-2 family that integrate into tRNA(Lys) or tRNA(Gly) genes represent hotspots of inter- and intraclonal genomic diversity. The individual islands differ in their repertoire of metabolic genes that make a large contribution to the pangenome. In order to unravel intraclonal diversity of P. aeruginosa, the genomes of two members of the PA14 clonal complex from diverse habitats and geographic origin were compared. The genome sequences differed by less than 0.01% from each other. One hundred ninety-eight of the 231 single nucleotide substitutions (SNPs) were non-randomly distributed in the genome. Non-synonymous SNPs were mainly found in an integrated Pf1-like phage and in genes involved in transcriptional regulation, membrane and extracellular constituents, transport, and secretion. In summary, P. aeruginosa is endowed with a highly conserved core genome of low sequence diversity and a highly variable accessory genome that communicates with other pseudomonads and genera via horizontal gene transfer.

Keywords: Pseudomonas aeruginosa; accessory genome; clonal complex; core genome; genome; genomic island; oligonucleotide signature.

Figure 1

Genome atlas representations of G + C content, tetranucleotide parameters and overrepresented 8- to 14mers in P. aeruginosa LESB58 (Davenport et al., 2009). Increasing divergence from average (up to an extreme value at ±3 SD) is indicated by progressively darker colors. G + C content and the three tetranucleotide parameters are plotted on the innermost four rings. Distance (second innermost circle) is the distance between global and local sliding window tetranucleotide patterns, pattern skew (third innermost circle) is the distance between tetranucleotide rankings on direct and reverse strands, and oligonucleotide variance (fourth innermost circle) is the numerical variance of oligomers, where a lower value indicates tetramer usage is more highly restricted (for example in repeat regions). Rings 5 (χ² threshold 3000) and 6 (χ² threshold 7000) display the number of bases occupied by overrepresented 8- to 14mers in a certain region, with overlaps only counted once, as a percentage. The outermost ring shows the difference (in classes) between a tetranucleotide parameter, oligonucleotide variance, and the 8- to 14mers in ring 5. Figures were created with JCircleGraph. Letters at the outermost ring indicate the regions of the six identified prophages (a–f) and five genomic islands (g–k; Winstanley et al., 2009).

Figure 2

The most overrepresented 8- 14 bp oligomers in P. aeruginosa LESB58 sorted by decreasing χ² values. The genome position of each oligo is plotted on the y-axis. A black dot is printed where an oligonucleotide occurs in non-coding regions and a green dot where an oligonucleotide occurs in coding regions. The figure was created with the program OligoViz (Davenport et al., 2009). The majority of the overrepresented 8–14 bp oligomers is located in coding sequences distributed all over the genome; only in the few cases of white vertical lines the respective oligonucleotide clusters in a few genome positions. Horizontal white lines indicate regions with an atypical oligonucleotide usage that lack these strain- or taxon-specific words and represent those parts of the accessory genome that are most foreign from the core.

Figure 3

Transposition of core genome DNA in LESB58. The genomic region with different core genome architecture is shown for strains PAO1 and LESB58. One hundred thirty-seven kbp of DNA (green) are located upstream of other core genome DNA blocks (gray) in LESB58 while occurring downstream of them in PAO1 (and other genomes). Surrounding core genome DNA arranged collinearly in both strains is shown in black, strain-specific insertions are represented by white areas. Genome coordinates of the borders of the core genome DNA blocks and numbers of the ORFs within are given for both strains. Accessory DNA blocks are described by the RGP number (see Table 2).

Figure 4

Conserved genes in pKLC102-/PAGI-2-like genomic islands. PAGI-2 (Larbig et al., 2002) and pKLC102 (Klockgether et al., 2004) were chosen as representatives for the respective subtypes among the pKLC102-/PAGI-2 family. The annotated ORFs are labeled according to their conservation. ORFs appearing in all P. aeruginosa islands of this family (“backbone genes”) are shown in black. ORFs conserved within one of the subtypes are colored in gray. White blocks represent ORFs specific for the single islands (“individual cargo”). Intergenic regions (igr) marked with an asterisk indicate loci with no ORF annotated for pKLC102 but for the highly homologous sequences in other islands from this subtype. Please note that ORF C105 of PAGI-2 is homologous to DNA in pKLC102 described as a part of the replication origin oriV of this element. The other part of oriV containing 16 57 bp repeats (Klockgether et al., 2004) is not conserved among the island family, not even in other islands from the pKLC102 subtype.

Figure 5

The P. aeruginosa pangenome. The extent of the P. aeruginosa core- and pan-genome is shown as a stepwise development going along with the availability of complete genome sequences. The numbers at the lower branch give the amount of genes identified as best reciprocal blast hits in the indicated genomes (core genome). Numbers of the upper branch describe amount of genes making up the pangenome. For each genome the number of genes are added that are neither ortho- nor paralogs of genes from the existing pool.

Figure 6

Intraclonal SNP diversity of the P. aeruginosa PA14 clone: distribution of nearest SNP neighbors in the RN3 genome. Mapping of the RN3 genome onto the PA14 genome uncovered 231 SNPs. The figure depicts the genomic distribution of the distance between two adjacent SNPs (nearest neighbors). The red graphs show the observed distribution that is compared with a random genomic distribution of the same number of 231 SNPs (blue graphs, one-dimensional random walk statistics). The two semilogarithmic plots visualize the deviation from a random distribution at either a global scale (insert) or with focus on the hotspots of sequence diversity (large figure).