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The overall architecture of IncP-1 plasmids is very conserved in that the accessory genes are typically located in one or two specific regions: between oriV and trfA and between the tra and trb operons. Various hypotheses have been formulated to explain this, but none have been tested experimentally. We investigated whether this structural similarity is due to region-specific transposition alone or also is reliant on selection for plasmids with insertions limited to these two regions. We first examined the transposition of Tn21Km into IncP-1[beta] plasmid pBP136 and found that most Tn21Km insertions (67%) were located around oriV. A similar experiment using the oriV region of IncP-1[beta] plasmid pUO1 confirmed these results. We then tested the transferability, stability, and fitness cost of different pBP136 derivatives to determine if impairment of these key plasmid characters explained the conserved plasmid architecture. Most of the pBP136 derivatives with insertions in transfer genes were no longer transferable. The plasmids with insertions in the oriV-trfA and tra-trb regions were more stable than other plasmid variants, and one of these also showed a significantly lower fitness cost. In addition, our detailed sequence analysis of IncP-1 plasmids showed that Tn402/5053-like transposons are situated predominantly between the tra and trb operons and close to the putative resolution site for the ParA resolvase, a potential hot spot for those transposons. Our study presents the first empirical evidence that region-specific insertion of transposons in combination with selection for transferable and stable plasmids explains the structural similarity of IncP-1 plasmids.

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (Ince-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra-and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.

The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1[beta] plasmid R751. Comparison of pUO1 with three other IncP-l[beta] plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.