Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product.

Abstract

PT181 is a naturally occurring 4.5 kbase Staphylococcus aureus plasmid encoding resistance to tetracycline. The plasmid has a copy number of .apprx. 20/cell; a mutant, cop-608, that has a copy number of 800-1000 was isolated. A cell-free extract was developed that carries out complete replication of this plasmid. Extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pT181. In an extract from which endogenous DNA has been removed, DNA synthesis is dependent upon the addition of exogenous plasmid DNA. The replication system is specific for pT181 and related plasmids but it is inactive with other S. aureus plasmids. pT181 DNA does not replicate in extracts made from plasmid-negative strains or strains containing other plasmids. A specific plasmid-encoded substance may be required for the replication of pT181 DNA.