IDENTIFICATION OF PLASMIDS IN ACTINOBACILLUS-ACTINOMYCETEMCOMITANS AND CONSTRUCTION OF INTERGENERIC SHUTTLE PLASMIDS

A collection of 39 isolates of Actinobacillus actinomycetemcomitans, o btained from laboratories located in 5 different geographical regions of the United States, was examined for the presence of plasmid DNA. On ly 2 of the strains examined, designated VT736 and VT745, harbored det ectable plasmids. Strain VT736 contained a 1.9 kb plasmid species (pVT 736-1) and a larger (> 30 kb) species (pVT736-2). Both plasmids were d etected in the covalently closed circular DNA fraction of dye buoyant density gradients. However, only the smaller plasmid was observed in a garose gels containing plasmid-enriched cell lysates prepared by a rap id screening procedure. Strain VT745 contained a single, 24 kb, plasmi d (pVT745) that was observed consistently in plasmid-enriched lysates, as well as in the plasmid band of dye buoyant density gradients. A re striction endonuclease map of pVT736-1 was constructed. The plasmid co ntained one site each for the enzymes HincII, KpnI and XhoI, located 6 00 to 700 bp from each other on the pVT736-1 map. HincII-digested pVT7 36-1 DNA could not be cloned in Escherichia coli. However, intact pVT7 36-1 digested with KpnI or XhoI could be cloned in E coli on pUC19 or pGEM7Zf(-), respectively. KpnI-digested pVT736-1 was cloned in both or ientations on pUC19, but XhoI-digested pVT736-1 was clonable in only o ne orientation on pGEM7Zf(-). Each of the 3 types of chimeric plasmid constructs provided a potential A. actinomycetemcomitans/E coli shuttl e plasmid for the development of a genetic transfer system in A. actin omycetemcomitans.