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Genetics and Molecular Biology |
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK1
Faculty of Biology, Department of Microbiology, Belarus State University, Scorina Av. 4, Minsk 220080, Belarus2
Author for correspondence: Christopher M. Thomas. Tel: +44 121 414 5903. Fax: +44 121 414 5925. e-mail: c.m.thomas{at}bham.ac.uk
Little is known of the transfer and maintenance machinery of the IncP-9 plasmids, which are found in Pseudomonas spp. and include both degradative and resistance plasmids. One such plasmid, pM3, which confers resistance to streptomycin and tetracycline, was found repeatedly in Pseudomonas species from numerous locations in Belarus. pM3 has a broad host range, but is unable to replicate in enterobacteria at 37 °C and above. A mini derivative, pMT2, was constructed by partial PstI digestion and ligation with a fragment encoding KmR. The complete sequence of pMT2 was determined. Analysis of its 8526 bp of pM3 DNA revealed several ORFs whose predicted polypeptide products were found to have similarity to previously analysed proteins involved in plasmid replication (rep gene), transfer (mpf; mating-pair formation gene) and stable maintenance (par, mrs genes). The organization of these genes showed similarity to several plasmid systems including the Ti and pSYM plasmids as well as IncP-1 plasmids. Subcloning narrowed down the region required for replication, and identified the putative rep gene and putative par promoter region as able to express incompatibility. rep deletion mutants were lost from the cell line, and expression of the rep gene was shown to be controlled by negative autoregulation. A pMT2 derivative with an insertion between the rep and par genes showed very weak, if any, ability to replicate autonomously, suggesting that plasmid maintenance may depend on a close interaction of rep and par functions.
Keywords: IncP-9 plasmids, broad-host-range replicon, plasmid incompatibility, antibiotic resistance, Pseudomonas putida
The GenBank accession number for the sequence reported in this paper is AF078924.
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