INACTIVATION OF THE REPLICATION-TERMINATION SYSTEM AFFECTS THE REPLICATION MODE AND CAUSES UNSTABLE MAINTENANCE OF PLASMID R1

Two so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional p artition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2 bp required for Tus binding also caused u nstable plasmid maintenance, whereas no effects on stability were obse rved when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA ge ne did not increase plasmid stability, suggesting that the multimeriza tion was not mediated by RecA. Plasmid DNA was isolated from the tus s train carrying plasmid pOU47 and from a wild-type strain carrying pOU4 7 in which the right Ter site had been inactivated; in both cases, ele ctron microscopy revealed the presence of multimers as well as rolling -circle structures with double-stranded tails. Thus, the right Ter sit e in plasmid R1 appears to stabilize the plasmid by preventing multime rization and shifts from theta to rolling-circle replication.