Abstract
The bacteriocinogenic plasmid Clo DF13 can be isolated from Escherichia coli cells and minicells as a complex of Clo DF13 DNA and one plasmid-specific protein, cloacin DF13 (Veltkamp et al., 1975). The construction of a physical map of Clo DF13 DNA on the basis of specific cleavage of Clo DF13 DNA by HindII restriction endonuclease (from Hemophilus influenzae), allowed us to determine the Clo DF13 DNA region(s) involved in the binding of cloacin DF13. It turned out that cloacin DF13 does not bind at random but to a specific Clo DF13 DNA region located on one of the HindII-generated Clo DF13 fragments. Circular Clo DF13 DNA can be cleaved under suitable conditions by in vivo as well as in vitro synthesized cloacin DF13 into linear DNA molecules of full Clo DF13 genome length. Cloacin DF13, which binds to supercoiled and open circular Clo DF13 DNA, remains bound to the DNA after cloacin-induced conversion of the circular form to the linear form. By using BamH-I restriction enzyme (from Bacillus amyloliquefaciens) it was demonstrated that cloacin DF13 acts on a very specific Clo DF13 DNA region, probably at one specific site, which is located on the BamH-I-generated Clo DF13 DNA fragment B. The optimal conditions for the endonuclease activity of cloacin DF13 are described. About 60% of the cloacin-generated linear Clo DF13 DNA molecules can be recircularized in vitro, indicating that cloacin DF13 makes two staggered, single-stranded endonucleolytic cuts in duplex Clo DF13 DNA. The binding as well as the endonucleolytic action of cloacin DF13 on Clo DF13 can be abolished by another Clo DF13 plasmid-specific protein, namely the immunity protein. Clo DF13 immunity protein itself has no affinity for Clo DF13 DNA but neutralizes the action of cloacin DF13 by forming a complex with this protein. The possible role of cloacin DF13 and Clo DF13 immunity protein in the replication of Clo DF13 plasmid DNA is discussed.
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