Role of CIS in Replication of an IncB Plasmid

Replication of the IncB plasmid pMU720 requires the synthesis of the cis-acting RepA protein and the presence of two DNA elements, ori and CIS. CIS is the 166-bp sequence separating the RepA coding sequence from ori. To investigate how this organization of the pMU720 replicon contributes to the mechanism of initiation of replication, mutations in the sequence and/or the length of CIS were introduced into the CIS region and their effects on the efficiency of replication of the pMU720 replicon in vivo was determined. The CIS region was found to be composed of two domains. The repA-proximal domain, which showed strong transcription termination activity, could be replaced by equivalent sequences from I-complex and IncL/M plasmids, whose replicons are organized in the same fashion as pMU720. Replacement by a trpA transcription terminator afforded only partial replication activity. The repA-distal domain was shown to be a spacer whose role was to position sequence(s) within ori on the correct face of the DNA helix vis-à-vis the repA-proximal portion of CIS. A model for the loading of RepA protein onto ori is discussed.