IDENTIFICATION OF A MAINTENANCE SYSTEM ON ROLLING CIRCLE REPLICATING PLASMID PVT736-1

Distribution of plasmid molecules to the two daughter cells at cell di vision is of major importance for their stable inheritance. Several me chanisms that control equipartitioning of low-copy-number plasmids hav e been described in molecular terms. However, no homologous or analogo us systems have been identified for intermediate or high-copy-number p lasmids, including rolling circle replicating (RCR) plasmids. It has b een suggested that distribution of such plasmids at cell division reli es solely on random segregation. Plasmid pVT736-1 is a 2kb RCR plasmid that was isolated from the Gram-negative capnophilic coccobacillus Ac tinobacillus actinomycetemcomitans. The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stab ility. An operon that consists of two genes (orf3 and orf2) is followe d by a putative cis-acting site that contains an integration host fact or (IHF) binding site, flanked by several repeats. Mutations in orf 2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or a nalogues of the pVT736-1 stabilization system have been detected on nu merous plasmid and bacterial genomes.