The replication and stable-inheritance functions of IncP-9 plasmid pM3

Little is known of the transfer and maintenance machinery of the IncP-9 pla smids, which are found in Pseudomonas spp. and include both degradative and resistance plasmids. One such plasmid, pM3, which confers resistance to st reptomycin and tetracycline, was found repeatedly in Pseudomonas species fr om numerous locations in Belarus. pM3 has a broad host range, but is unable to replicate in enterobacteria at 37 degrees C and above. A mini derivativ e, pMT2, was constructed by partial PstI digestion and ligation with a frag ment encoding Km(R). The complete sequence of pMT2 was determined. Analysis of its 8526 bp of pM3 DNA revealed several ORFs whose predicted polypeptid e products were found to have similarity to previously analysed proteins in volved in plasmid replication (rep gene), transfer (mpf; mating-pair format ion gene) and stable maintenance (par, mrs genes). The organization of thes e genes showed similarity to several plasmid systems including the Ti and p SYM plasmids as well as IncP-1 plasmids. Subcloning narrowed down the regio n required for replication, and identified the putative rep gene and putati ve par promoter region as able to express incompatibility. rep deletion mut ants 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 ins ertion between the rep and par genes showed very weak, if any, ability to r eplicate autonomously, suggesting that plasmid maintenance may depend on a close interaction of rep and par functions.