Nucleotide sequence and analysis of conjugative plasmid pVT745.

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.

Identification of a maintenance system on rolling circle replicating plasmid pVT736-1. dgalli@iusd.iupui.edu.

Distribution of plasmid molecules to the two daughter cells at cell division is of major importance for their stable inheritance. Several mechanisms that control equipartitioning of low-copy-number plasmids have been described in molecular terms. However, no homologous or analogous systems have been identified for intermediate or high-copy-number plasmids, including rolling circle replicating (RCR) plasmids. It has been suggested that distribution of such plasmids at cell division relies solely on random segregation. Plasmid pVT736-1 is a 2 kb RCR plasmid that was isolated from the Gram-negative capnophilic coccobacillus Actinobacillus actinomycetemcomitans. The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stability. An operon that consists of two genes (orf3 and orf2) is followed by a putative cis-acting site that contains an integration host factor (IHF) binding site, flanked by several repeats. Mutations in orf2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or analogues of the pVT736-1 stabilization system have been detected on numerous plasmid and bacterial genomes.

Structural and segregational stability of various replicons in Actinobacillus actinomycetemcomitans.

Few vectors suitable for cloning in Actinobacillus actinomycetemcomitans, a periodontal pathogen, have been described. These plasmids were based either on the native A. actinomycetemcomitans replicon, pVT736-1, or derivatives of the IncP and IncQ family of plasmids. Their usefulness as cloning vectors is limited because of instability or size. Therefore, the ability of additional replicons to function in A. actinomycetemcomitans was evaluated. Derivatives of p15A, ColE1/f1, and pWV01 transformed A. actinomycetemcomitans efficiently and exhibited no structural instability in the new host. The stable maintenance of A. actinomycetemcomitans-specific DNA fragments inserted into the p15A derivative, pDMG4, demonstrated the ability of this plasmid to function as a cloning vector. In addition, pVT736-1 was used to clone selectable markers directly into A. actinomycetemcomitans. These constructs were structurally and segregationally stable.

Transcriptional analysis of rolling circle replicating plasmid pVT736-1: evidence for replication control by antisense RNA.

Several plasmids have been described in Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus. Recently, the nucleotide sequence of pVT736-1, a cryptic plasmid of A. actinomycetemcomitans VT736, was determined. This plasmid possesses all the features necessary for rolling circle replication. The present study involved a transcriptional analysis of pVT736-1. Results of Northern (RNA) blot analyses and primer extension studies indicated that the two open reading frames identified in pVT736-1 are each preceded by at least one promoter. Expression of these promoters varied with growth phase. In addition, an antisense RNA (Cop RNA) appeared to control the synthesis of the putative replication protein. To our knowledge, this is the first rolling circle replicating plasmid isolated from a gram-negative organism that has been subjected to such detailed analysis.

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.