Complete nucleotide sequence of TOL plasmid pDK1 provides evidence for evolutionary history of IncP-7 catabolic plasmids

Abstract

To understand the mechanisms for structural diversification of Pseudomonas-derived toluene-catabolic (TOL) plasmids, the complete sequence of a self-transmissible plasmid pDK1 with a size of 128,921 bp from Pseudomonas putida HS1 was determined. Comparative analysis revealed that (i) pDK1 consisted of a 75.6-kb IncP-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pDK1 were highly similar to those of MOB(H) group of mobilizable plasmids, and (iii) the toluene-catabolic (xyl) gene clusters of pDK1 were derived through homologous recombination, transposition, and site-specific recombination from the xyl gene clusters homologous to another TOL plasmid, pWW53. The minireplicons of pDK1 and its related IncP-7 plasmids, pWW53 and pCAR1, that contain replication and partition genes were maintained in all of six Pseudomonas strains tested, but not in alpha- or betaproteobacterial strains. The recipient host range of conjugative transfer of pDK1 was, however, limited to two Pseudomonas strains. These results indicate that IncP-7 plasmids are essentially narrow-host-range and self-transmissible plasmids that encode MOB(H) group-related transfer functions and that the host range of IncP-7-specified conjugative transfer was, unlike the situation in other well-known plasmids, narrower than that of its replication.

FIG. 1.

Circular map of pDK1. CDSs are shown as boxes inside or outside the circle with gene names or ofn numbers. Color indicates proposed functions of gene products: green, replication, partition, and DNA-processing; blue, transposition and site-specific recombination; purple, conjugative DNA transfer; red, degradation of toluene/xylenes; yellow, other known or unknown functions; and gray, gene remnants. Transposon-derived segments are represented by blue arcs. Terminal IRs and res regions of class II transposons are indicated as filled triangles and open circles, respectively. The segments highly homologous to those in pWW53, pND6-1, and pCAR1 (revealed by pairwise BLASTn analysis) are indicated by black, gray, and white arcs, respectively.

FIG. 2.

Schematic representation of insertions and deletions in pDK1, pWW53, pCAR1, and pND6-1. A proposed IncP-7 backbone is represented by a gray bar with landmark genes. pDK1- and pWW53-specific inserts are shown above the backbone, while pCAR1- and pND6-1-specific inserts are shown below the backbone. Genes for the proteins with ambiguous or hypothetical functions in pDK1, pWW53, and pCAR1 are designated ofn, ww, and ORF, respectively. Filled arrowheads indicate terminal IRs of class II transposons. The replication/partition/regulation region (the 14-kb segment from tus to hns) and conjugative DNA transfer region (the 45-kb segment from trhN to ofn74) are indicated.

FIG. 3.

Gene organization in replication/partition region of the IncP-7 plasmid. Depicted is the pDK1 segment from coordinates 22283 to 8760. Coding sequences are represented by pentagons. Arrows inserted between parA and parB indicate the 17-bp palindromic motifs. Gene products required for stable maintenance of the pCAR1 replicon in P. putida (54) are indicated in gray. The repB and hns genes in pDK1 (hatched pentagons) are inactivated by IS1162 insertion and nonsense mutations, respectively (see text for details). The identities of gene products of other IncP-7 plasmids with those of pDK1 are shown below the gene names. To calculate the identities for RepB and H-NS, their respective wild-type proteins of pDK1 are predicted by the in silico removal of mutations (the insertion mutation of IS1162 in repB and the nonsense mutation at the 38th codon in hns in pDK1). NA, not available due to lack of gene homologs in pND6-1. Putative promoters and Rho-independent terminators are indicated by flags and pins, respectively. Two HindIII sites used for construction of mini-IncP-7 replicons, pMM67 (mini-pCAR1), pMM68 (mini-pDK1), and pHY118 (mini-pWW53) (73), are depicted.

FIG. 4.

Comparison of pDK1 and pWW53. (A) Similarity in xyl-containing regions of pDK1 and pWW53. Results of BLAST analysis are shown. Pentagons indicate coding sequences. The two xylS genes (xylS1 and xylS3) and a truncated xylS gene (xylS2) are represented by filled pentagons. (B) Sequence comparison among Tn21-related res regions. Dots indicate nucleotides identical to those in Tn21. Boxed are the three binding sites of TnpR in the Tn21 res region (46) and putatively in the res regions in other Tn21-related transposons. The center of site I, where the staggered-cut and subsequent strand exchange takes place, is shaded.

FIG. 5.

Proposed model for generation of a pDK1-type xyl gene cluster from a pWW53-type cluster: event 1, transposition of TnAtcArs into the pDK1 ancestor; event 2, homology-dependent intracellular inversion between xylS1 and xylS3; event 3, TnpR-mediated site-specific resolution between res2 and the res region of the TnAtcArs-like transposon. The order of events 1 and 2 is unclear. See text for details.