Abstract
The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 replication specifically requires the plasmid-encoded repC protein. An in vitro system has been shown to carry out semiconservative replication of pT181 and its derivative plasmids (Khan, S A., Carleton, S. M., and Novick, R. P. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4902-4906). We have used this replication assay to isolate repC protein, which was purified to near homogeneity. The repC gene was cloned into the pKJB825 plasmid that contains the phage lambda temperature-sensitive repressor gene, cI857, and the rightward promoter, PR. Upon temperature induction, Escherichia coli clones containing the recombinant plasmid overproduced repC protein, which was purified in significant quantities. The molecular weight of repC protein under denaturing conditions is 38,000, which is consistent with the size predicted from the DNA sequence data. Presence of repC protein was absolutely essential for the initiation of replication of pT181 and its derivatives in vitro.
Highlights
Upon temperature ing conditions is 38,000, which is consistent with the induction, E. coli strains containing pSK184 overproduced size predicted from the DNA sequence data
The of repC protein was absolutely essential for the initia- molecular weight of repC protein as determined by mobility tion of replication of pT181 and its derivatives in vitro. in SDS-polyacrylamide gels was found to be approximately
The cop608 plasmid is similar to pT181 except that ithas a 180-bp deletion within the repC leader sequence that is involved in the inhibition of repC protein synthesis [21, 22, 40]
Summary
Cloning of the repC Gene Downstream from the X PRPromoter-The coding sequence for repC protein on pT181DNA was identified earlier [7, 22]. The 1251-bpRsaI B fragment from cop608, containing the repC gene [22], was cloned into the HincII site of pUC7 and transformed into E. coli JM83. The resulting plasmid, pSK179 (Fig. l ) , was identified and a 1.3-kbEcoRI fragment containingthe repC gene wasisolated. This fragment was ligated into the EcoRI site of pKJB825 and transformed into E . Purification of repC Protein-Overproduction of repC protein by strain MBZ(pSK184) resulted from enhanced transcription of the repCgene from the phage X PRpromoter, induced by temperature inactivation of the product of the FIG.. The orientation of the repCgene in pSK184 was determined by restriction enzyme analysis
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