Region-Specific Insertion of Transposons in Combination with Selection for High Plasmid Transferability and Stability Accounts for the Structural Similarity of IncP-1 Plasmids

<jats:title>ABSTRACT</jats:title><jats:p>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<jats:italic>oriV</jats:italic>and<jats:italic>trfA</jats:italic>and between the<jats:italic>tra</jats:italic>and<jats:italic>trb</jats:italic>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 Tn<jats:italic>21</jats:italic>Km into IncP-1β plasmid pBP136 and found that most Tn<jats:italic>21</jats:italic>Km insertions (67%) were located around<jats:italic>oriV</jats:italic>. A similar experiment using the<jats:italic>oriV</jats:italic>region of IncP-1β 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<jats:italic>oriV</jats:italic>-<jats:italic>trfA</jats:italic>and<jats:italic>tra-trb</jats:italic>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 Tn<jats:italic>402</jats:italic>/<jats:italic>5053</jats:italic>-like transposons are situated predominantly between the<jats:italic>tra</jats:italic>and<jats:italic>trb</jats:italic>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.</jats:p>