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.

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 SC and above. A mini derivative, pMT2, was constructed by partial PstI digestion and ligation with a fragment encoding Km R . 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.

IncP-9 plasmids are best known as the vehicles for spreading biodegradation functions among Pseudomonas species but can also carry resistance determinants. New PCR primer systems targeting different replicon-specific regions were designed to allow detection of IncP-9 plasmids. Their specificity was checked against a range of IncP-9 plasmids as well as representatives of incompatibility groups IncFI, IncFII, IncN, IncQ, IncP-1alpha, IncP-1beta, IncP-2, IncP-7, IncP-13, IncW, IncU, IncX and IncZ. Products obtained for plasmids assigned to IncP-9 group by traditional incompatibility testing varied in size and restriction pattern suggesting diversity in the 'core' sequence among related replicons. Specific primer pairs were applied to community DNA extracted from a range of environments including those subject to strong selective pressure, caused by antibiotics, metals and organic pollutants. Abundant products were observed in manure and sewage, independently of the presence of antibiotics and metals, but could also be detected in coastal water and streptomycin-treated soil. Community DNA from faeces of piglets treated and non-treated with Zn gave particularly strong PCR product with IncP-9 rep primers. Therefore, an attempt was made to isolate bacteria carrying the IncP-9-like plasmids, but this was not successful. The results of application of these newly designed primer pairs to plasmid isolates as well as community DNA indicate that the IncP-9-related plasmids are a diverse family prevalent in various environments and widely distributed geographically.

Ability of IncP-9 plasmid pM3 to replicate in SummaryIncP-9 plasmids are common in Pseudomonas species and can be transferred to other Gram-negative eubacteria but tend not to be stably maintained outside their natural host genus. A 1.3 kb ori V-rep fragment from IncP-9 plasmid pM3 was sufficient for autonomous replication in Pseudomonas putida but not in Escherichia coli . Replication of ori V-rep in E. coli was restored when additional rep was provided in trans , suggesting that the replication defect resulted from insufficient rep expression from its natural promoter. A promoter deficiency in E. coli was confirmed by reporter gene assays, transcriptional start point mapping and mutation of the promoter recognition elements. Dissection of the pM3 mini-replicon, pMT2, showed that this replication deficiency in E. coli is suppressed by additional determinants from its par operon: ParB, which can be supplied in trans , and its target, the par operon promoter, required in cis to ori V-rep . We propose that ParB binding to its target either changes plasmid DNA and thus promoter conformation or by spreading or looping contacts RNAP at the rep promoter so that rep expression is sufficient to activate ori V.

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