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.
Department of Microbiology, University of Texas Health Science Center at San Antonio 78284.
D M
DM
Galli
D J
DJ
LeBlanc
D000188:Actinobacillus / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010957:Plasmids; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C088222:PVT736-1 replication protein, Actinobacillus actinomycetemcomitans