Isolation and characterization of deletion derivatives of pDL282, an Actinobacillus actinomycetemcomitans/Escherichia coli shuttle plasmid.

Recent reports have described the construction of several shuttle plasmids (D.J. LeBlanc, L. L. Lee, A. Al-Jaibat, P. K. Sreenivasan, and P. M. Fives-Taylor, Oral Micro. Immunol. (1993) 8, 94-99) and the development of an efficient transformation system for Actinobacillus actinomycetemcomitans (P. K. Sreenivasan, D. J. LeBlanc, L. L. Lee, and P. M. Fives-Taylor, Infect. Immun. (1991) 59, 4621-4627), a gram-negative bacterium implicated in human periodontal disease. This report presents results from further studies on pDL282, an A. actinomycetemcomitans-Escherichia coli shuttle plasmid. A. actinomycetemcomitans containing pDL282 lost the plasmid at a rapid rate when cultured in antibiotic free medium. Intact pDL282 was maintained for 20 generations or more when the host cells were grown in the presence of ampicillin, or ampicillin plus spectinomycin. However, prolonged incubation in the presence of spectinomycin only resulted in the emergence of one or the other of two unique deletion derivatives of pDL282, designated pPK1 and pPK2. Whereas A. actinomycetemcomitans was efficiently transformed with pDL282 (5.7 kb), pPK1 (3.6 kb), and pPK2 (2.5 kb), E. coli was transformed only by the two largest species. Like the parent molecule, pPK1 and pPK2 were rapidly lost from A. actinomycetemcomitans hosts in the absence of antibiotic selection. Neither pPK1 nor pPK2 suffered any further deletions following prolonged cultivation in the presence of spectinomycin. The minimal replicon of pVT736-1, the A. actinomycetemcomitans-derived plasmid component of pDL282, was located within a 1400 bp DNA fragment of pPK1 and pPK2.(ABSTRACT TRUNCATED AT 250 WORDS)