Abstract
IncP-9 plasmids are important vehicles for degradation and resistance genes that contribute to the adaptability of Pseudomonas species in a variety of natural habitats. The three completely sequenced IncP-9 plasmids, pWW0, pDTG1 and NAH7, show extensive homology in replication, partitioning and transfer loci (an ∼25 kb region) and to a lesser extent in the remaining backbone segments. We used PCR, DNA sequencing, hybridization and phylogenetic analyses to investigate the genetic diversity of 30 IncP-9 plasmids as well as the possibility of recombination between plasmids belonging to this family. Phylogenetic analysis of rep and oriV sequences revealed nine plasmid subgroups with 7–35 % divergence between them. Only one phenotypic character was normally associated with each subgroup, except for the IncP-9β cluster, which included naphthalene- and toluene-degradation plasmids. The PCR and hybridization analysis using pWW0- and pDTG1-specific primers and probes targeting selected backbone loci showed that members of different IncP-9 subgroups have considerable similarity in their overall organization, supporting the existence of a conserved ancestral IncP-9 sequence. The results suggested that some IncP-9 plasmids are the product of recombination between plasmids of different IncP-9 subgroups but demonstrated clearly that insertion of degradative transposons has occurred on multiple occasions, indicating that association of this phenotype with these plasmids is not simply the result of divergent evolution from a single successful ancestral degradative plasmid.
Highlights
As agents of horizontal gene transfer (HGT), plasmids contribute considerably to bacterial evolution and adaptation (Thomas, 2000a)
Bacteria were propagated in Luria broth or M9 minimal medium, supplemented with 1.5 % agar when required (Sambrook & Russell, 2001)
To select for catabolic plasmids, bacteria were maintained on M9 lacking glucose but supplemented with an appropriate source of carbon and energy such as naphthalene [0.05–0.1 g naphthalene (Sigma) placed on the lid of an inverted Petri dish], caprolactam and toluene (0.1–0.15 % and 5 mM added to molten agar before pouring, respectively)
Summary
As agents of horizontal gene transfer (HGT), plasmids contribute considerably to bacterial evolution and adaptation (Thomas, 2000a). PCR primers that target the basic replicon functions of IncP-9 have facilitated studies on the diversity and prevalence of naturally occurring IncP-9 plasmids (Greated & Thomas, 1999; Krasowiak et al, 2002). The genetic diversity of a limited number of phenotypically different IncP-9 plasmids (Krasowiak et al, 2002) and large collections of IncP-9 Nah replicons (Izmalkova et al, 2006; Leuchuk et al, 2006) have been evaluated by RFLP analysis of replication/maintenance loci, selected nah determinants or complete plasmid genomes. Subsequent analysis of selected backbone loci indicated that phylogenetically distant IncP-9 plasmids must retain many common genes, while modules encoding similar phenotypes have been inserted at different sites in the conserved plasmid backbone, indicating multiple independent acquisitions of these functions
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