Identification of mutations affecting replication control of plasmid Clo DF13

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Abstract
The bacteriocinogenic plasmid Clo-DF13, originally isolated from Enterobacter 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 and Clo-DF13 are located 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. Transcription in the opposite direction results in a small transcript of .apprx. 100 nucleotides (RNA-100) for both Col-E1 and Clo-DF13. Apparently Col-E1 RNA-100 acts as a negative control element for the initiation of replication. Single base transitions in the RNA-100 cistron of Clo-DF13 can result in a nonconditional increase in plasmid copy-number. Sequence analysis 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.