Identification of mutations affecting replication control of plasmid Clo DF13

Abstract

The bacteriocinogenic plasmid Clo DF13, originally isolated from Escherichia cloacae, is stably maintained in Escherichia coli to the extent of about 10 copies per cell. Its replication resembles that of many other small, multicopy plasmids; plasmid-encoded protein is not required but plasmid-specific genetic information is involved in regulation of replication as both conditional and nonconditional copy-number mutants of Clo DF13, and transcomplementable copy-number mutants of plasmid Col E1 have been described. The sequences essential for replication of Col E1 (refs 16, 17) and Clo DF13 (refs 18, 19) have been identified within a region surrounding the replication origin. Initiation of Col E1 replication is preceded by transcription of the origin region, providing the RNA primer at the origin. However, transcription in the opposite direction results in a small transcript of approximately 100 nucleotides (RNA-100) for both Col E1 (refs 21, 22) and Clo DF13 (ref. 23). Data suggest that Col E1 RNA-100 acts as a negative control element for the initiation of replication. We show here that single base transitions in the RNA-100 cistron of Clo DF13 can result in a nonconditional increase in plasmid copy-number. Also, sequence analysis has revealed that a specific base transition in a DNA region, apparently involved in both termination and initiation of transcription towards the replication origin, results in a thermosensitive plasmid copy-number.