Diversity of IncP-9 plasmids of Pseudomonas

1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 2 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142290, Russia 3 Genetics Department, Biology Faculty, Belarus State University, 6 Kurchatova St, Minsk 220064, Belarus 4 Julius Kühn Institute – Federal Research Centre for Cultivated Plants (JKI), Messeweg 11/12, 38104 Braunschweig, Germany Correspondence Christopher M. Thomas c.m.thomas{at}bham.ac.uk 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-9 β 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. Abbreviations: HGT, horizontal gene transfer Present address: Department of Biology, University of York, PO Box 373, York YO10 5YW, UK. Present address: Division of Immunity and Infection, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. The GenBank/EMBL/DDBJ accession numbers for the newly determined oriV and rep sequences of Pseudomonas strains examined in this study are EU499619–EU4