Iteron control of oriV function in IncP-1 plasmid RK2

Replication control of many plasmids is mediated by the balance between the positive and negative effects of Rep protein binding repeated sequences (iterons) associated with the replication origin, oriV. Negative control is thought to be mediated by dimeric Rep protein linking iterons in a process termed handcuffing. The wellstudied oriV region of RK2 contains 9 iterons arranged as a singleton (iteron 1), a group of 3 (iterons 2-4) and a group of 5 (iterons 5-9), but only iterons 5 to 9 are essential for replication. An additional iteron (iteron 10), oriented in the opposite direction, is also involved and reduces copy-number nearly two-fold. Since iterons 1 and 10 share an identical upstream hexamer (5' TTTCAT 3 ') it has been hypothesised that they form a TrfAmediated loop facilitated by their inverted orientation. Here we report that contrary to the hypothesis, flipping one or other so they are in direct orientation results in marginally lower rather than higher copy-number. In addition, following mutagenesis of the hexamer upstream of iteron 10, we report that the Logo for the hexamer upstream of the regulatory iterons (1 to 4 and 10) differs from that of the essential iterons, suggesting functional differences in their interaction with TrfA.

Automated summary

The replication control of plasmids is influenced by the interaction between Rep protein and repeated sequences known as iterons in the replication origin, oriV. Negative control is achieved through Rep protein handcuffing iterons. The well-studied oriV region of RK2 contains 9 iterons, but only iterons 5 to 9 are essential for replication. Flipping the orientation of iterons 1 and 10, which share the same upstream hexamer, does not increase but rather slightly decrease the copy-number. Mutagenesis of the hexamer upstream of iteron 10 suggests functional differences in the interaction with TrfA compared to the essential iterons.