CHARACTERIZATION OF PVT736-1, A ROLLING-CIRCLE PLASMID FROM THE GRAM-NEGATIVE BACTERIUM ACTINOBACILLUS-ACTINOMYCETEMCOMITANS

The presence of plasmid DNA in Actinobacillus actinomycetemcomitans has been reported. Recently, the construction of an Escherichia coli/A. actinomycetemcomitans shuttle vector, based on the A. actinomycetemcomitans-derived 2.0-kb plasmid pVT736-1, was described (D. J. LeBlanc, A. R. Abu-Al-Jaibat, P. K. Sreenivasan, and P. M. Fives-Taylor, Oral Microbiol. Immunol. (1993) 8, 94-99). The current study was initiated with the determination of the nucleotide sequence of pVT736-1, which revealed the presence of two open reading frames (ORFs) encoding proteins of 293 (ORF1) and 95 (ORF2) amino acids. Evidence that pVT736-1 replicates via a single-stranded (ss) intermediate included: (i) the presence of ssDNA in cells and in cell-free supernatant, (ii) the presence of conserved sequence motifs in the predicted ORF1 protein that are typical of initiator (Rep) proteins associated with replication by a rolling-circle mode, and (iii) 39% amino acid identity between the putative Rep proteins of pVT736-1 and Pf3, a filamentous ssDNA bacteriophage of Pseudomonas aeruginosa. A putative plus origin of replication in pVT736-1 was located upstream of ORF1, in a 200-bp region with potential for secondary hairpin structures. The identification in gram-negative bacteria of extrachromosomal DNA (other than bacteriophage) that replicates by a rolling-circle mode and shows no extensive homology to plasmids from gram-positive organisms is rather unique. (C) 1994 Academic Press, Inc.