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

Abstract

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.Actinobacillus actinomycetemcomitans is a gram-negative, capnophilic coccobacillus associated with various forms of periodontitis (34). The presence of plasmid DNA in A. actinomycetemcomitans has been reported previously (18,23). Characterization of these plasmids is important in terms of their possible carriage and dissemination of virulence genes and for the development of genetic and molecular tools applicable to A. actinomycetemcomitans. Recently, the nucleotide base sequence of pVT736-1, a cryptic plasmid of A. actinomycetemcomitans VT736, was determined (13). Computer analysis revealed the presence of two open reading frames encoding predicted proteins of 293 (ORF1) and 95 (ORF2) amino acids (13). Evidence that pVT736-1 replicates via a rolling circle mode included the presence of single-stranded DNA (ssDNA) in cells and in cell-free supernatant and the identification of conserved sequence motifs in the predicted ORF1 protein that are typical of initiator (Rep) proteins associated with this type of replication (13).Rolling circle replicating (RCR) plasmids have been identified primarily in gram-positive bacteria. Novick (21) has divided these plasmids into four groups, represented by pT181, pUB110/pC194, pLS1/pE194, and pSN2. To date, the regulation of replication has been well characterized for only two RCR plasmids, the staphylococcal plasmid pT181 (for a review, see reference 21) and the streptococcal plasmid pLS1 (6). Plasmid-encoded products control plasmid copy number in both cases: two antisense RNAs for pT181 (17) and an antisense RNA and a small repressor protein for pLS1 (6). The target of the antisense transcripts in both instances is the mRNA of the plasmid-specific initiator protein (Rep protein), but the mode of antisense action is different. Binding of the antisense RNA induces premature termination of transcription (attenuation) in the Rep RNA of pT181 (22), whereas the countertranscript inhibits translation of the target messenger of pLS1 by blocking the ribosome binding site (RBS) (6).Recently, a number of RCR plasmids have been reported in gram-negative bacteria (reviewed in references 7 and 27). With the exception of pVT736...

Identification of a maintenance system on rolling circle replicating plasmid pVT736‐1

“…4) are located on this segment. It has been shown that both genes form an operon (Galli and LeBlanc, 1995). The orf2 gene product is a trans-acting protein.…”

“…However, two specific transcripts were associated with orf2 : m2 (c. 0.8 kb) and m2Ј (c. 0.6 kb) (Galli and LeBlanc, 1995). Since m2 exceeded the length of orf2 by more than 0.5 kb, the sequence of pVT736-1 was reanalysed to search for the presence of additional open reading frames.…”

“…1). This resulted in the loss of the 5Ј end of orf2 and its ribosome-binding site, but left the putative promoter region P2Ј intact (Galli and LeBlanc, 1995). The deletion in pDMG3-2 was extended in both directions by exonuclease III digestion, which yielded pDMG3-3B.…”

“…Doublestranded DNA was shortened with exonuclease III (Gibco BRL) at room temperature, followed by digestion of the single-stranded tails with S1 nuclease (Gibco BRL) (Henikoff, 1987). Ligation (Sambrook et al, 1989), labelling (Galli and LeBlanc, 1995) and Southern transfer of DNA (Southern, 1975) and DNA-DNA hybridization conditions (Galli and Wirth, 1991) have been described previously. Transformations of E. coli and A. actinomycetemcomitans by electroporation were performed according to standard procedures (Fiedler and Wirth, 1988;Sreenivasan et al, 1991).…”

Bacterial plasmids: replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds

“…A number of small, high copy number plasmids from Gram-positive bacteria have been initially shown to replicate using the rolling circle mechanism of replication and later some plasmids of Gram-negative bacteria were identified that use this mechanism of replication (41, [146][147][148][149][150][151][152][153]. The molecular mechanisms of replication of plasmids pT181, pC221, pUB110, pC194 and pMV158 are the best known to date (43, 53, 154-158).…”