Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C

Abstract

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.

FIG. 1.

(a) Restriction map of plasmid pKLC102 (inner circle, EcoRI; outer circle, PvuII). The recombination site for chromosomal integration and the position of the insertion of integron TNCP23 in strain C are indicated. The thick arcs represent the tiling path of cosmids and gap-spanning PCR products utilized for sequencing pKLC102 DNA in the strain C chromosome. The darkly shaded area is absent in pKLK106 (see panel b). (b) Comparative restriction analysis of pKLC102 and pKLK106. (I) Separated HindIII, EcoRI, and PvuII restriction digests of cosmids pKSCC785 (lanes 1), pKSCC187 (lanes 2), pKSCC050 (lanes 3), and pKSCC867 (lanes 4). PCR, gap-spanning PCR product (undigested); λ, BstEII digest of λ DNA used as a size standard. (II) Southern blot of gel I, hybridized with plasmid pKLK106. The letters in gel I indicate bands with no or lower-than-expected hybridization signals due to DNA that is not represented in the pKLK106 probe. P, PAO1 DNA flanking the inserted pKLC102 in strain C; V, vector DNA; T, integron TNCP23; C (circled), pKLC102-specific DNA absent in pKLK106.

FIG. 2.

Map of tRNA^Lys-phnAB regions of strain K, PAO1, and C chromosomes. The tRNA^Lys sites are indicated by thick black bars. In clone K strains, the tRNA^Lys site can be used for reversible integration of plasmid pKLK106 (green triangle) (36). PAO1 carries an additional block (light gray triangle) at this site, comprising CDS PA0977 to PA0987. Strain C carries the gene island PAGI-4(C) at this position. Base pair counting starts after tRNA^Lys. Two small segments (dark gray) with ORFs PA0977 and PA0980 are homologous to PAO1 sequence; two larger areas (yellow and orange) are C specific. The blue arrows show PAO1 CDS and their counterparts in K and C; the yellow and orange arrows represent C-specific CDS in PAGI-4(C). The blue boxes represent truncated PAO1 CDS in strain C.

FIG. 3.

Gene map of pKLC102. The map is calibrated to the chromosomal integration attP site, marked by a flag. The leading strand was defined by colinearity with the P. aeruginosa PAO1 genome sequence. Predicted coding regions are shown by arrows indicating the direction of transcription. The genes are color coded according to their functional categories, as shown in the legend below the map. All genes carry identification numbers according to the CDS numbering in Table 3. Homologs in other microorganisms retrieved by a BLASTP search and identified gene names are highlighted beneath the corresponding CDS. oriV is the predicted origin of replication. The putative CDS within the origin of replication is shown by a dotted arrow. The syntenic CDS CP73 to CP81 that were subjected to cladistic analysis (Fig. 5) are marked by bent arrows.

FIG. 4.

Structure of the origin of replication of pKLC102. Identical sequences are indicated by the sizes of the symbols. Adjacent solid and open boxes represent palindromes; the arrows indicate the sequences of 16 consecutive direct repeats. The A+T-rich region is indicated by a horizontal black bar.

FIG. 5.

Circular domain similarity plot. The inner and outer circles represent 50 and 100% similarities, respectively. Plasmid coordinates are shown along the outer circle.

FIG. 6.

Inner opposite CDS of XerC integrases CL2ab, CP103ab, and Avin0928. The integrase genes and putative traI genes CL3, CP102, and Avin0927 located upstream of the integrases are shown by open arrows. The integration attachment sites downstream of the integrases are indicated by solid boxes. Identified inner ORFs (putative excisionases) are depicted by shaded arrows. The boxed sequences indicate the putative termination loops following the inner ORFs in pKLC102 and A. vinelandii.

FIG. 7.

Gene map of TNCP23. The map is calibrated to the site of integration into the chromosome of strain C. The leading strand was defined by colinearity with the P. aeruginosa PAO1 genome sequence. Predicted coding regions are shown by arrows indicating the direction of transcription. The genes are color coded according to their functional categories as shown in the legend below the map. All of the genes carry identification numbers according to the CDS numbering in Table 4, but the abbreviation Tn was used instead of TNCP due to space limitations. Gene names are highlighted beneath the corresponding CDS. oriV is the predicted origin of replication.

FIG. 8.

Evolution of P. aeruginosa strains linked to plasmid DNA. (a) Reversible integration of plasmid DNA into two possible sites of clone K strains. (b) Different forms of plasmid DNA in clone C strains. In subgroup SG17M, pKLC102 is found episomally and integrated into the genome at tRNA^Lys(2). Strain C5 apparently lost the pKLC102 DNA, while strain C2 harbors only the integrated form. In subgroup C, the integron TNCP23 inserted into chromosomally integrated pKLC102. Free plasmid is not detectable in subgroup C strains, indicating that TNCP23 prevented mobilization. TNCP23 is flanked by copies of IS6100. Intramolecular transposition of the left copy of IS6100-L is coupled with an inversion of the chromosomal region between the transposed copy and IS6100-L in some strains of subgroup C. For these strains C8, C9, C10, and C19, the tRNA^Lys(1) area is not shown.