Identification of plasmids in Actinobacillus actinomycetemcomitans and construction of intergeneric shuttle plasmids

Abstract

A collection of 39 isolates of Actinobacillus actinomycetemcomitans, obtained from laboratories located in 5 different geographical regions of the United States, was examined for the presence of plasmid DNA. Only 2 of the strains examined, designated VT736 and VT745, harbored detectable plasmids. Strain VT736 contained a 1.9 kb plasmid species (pVT736-1) and a larger ( > 30 kb) species (pVT736-2). Both plasmids were detected in the covalently closed circular DNA fraction of dye buoyant density gradients. However, only the smaller plasmid was observed in agarose gels containing plasmid-enriched cell lysates prepared by a rapid screening procedure. Strain VT745 contained a single, 24 kb, plasmid (pVT745) that was observed consistently in plasmid-enriched lysates, as well as in the plasmid band of dye buoyant density gradients. A restriction endonuclease map of pVT736-1 was constructed. The plasmid contained one site each for the enzymes HincII, KpnI and XhoI, located 600 to 700 bp from each other on the pVT736-1 map. HincII-digested pVT736-1 DNA could not be cloned in Escherichia coli. However, intact pVT736-1 digested with KpnI or XhoI could be cloned in E. coli on pUC19 or pGEM7Zf(-), respectively. KpnI-digested pVT736-1 was cloned in both orientations on pUC19, but XhoI-digested pVT736-1 was clonable in only one orientation on pGEM7Zf(-). Each of the 3 types of chimeric plasmid constructs provided a potential A. actinomycetemcomitans/E. coli shuttle plasmid for the development of a genetic transfer system in A. actinomycetemcomitans.