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. 1995 Aug;177(15):4474–4480. doi: 10.1128/jb.177.15.4474-4480.1995

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

D M Galli 1, D J Leblanc 1
PMCID: PMC177199  PMID: 7543479

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.

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Selected References

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  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Blomberg P., Wagner E. G., Nordström K. Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III. EMBO J. 1990 Jul;9(7):2331–2340. doi: 10.1002/j.1460-2075.1990.tb07405.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bläsi U., Nam K., Hartz D., Gold L., Young R. Dual translational initiation sites control function of the lambda S gene. EMBO J. 1989 Nov;8(11):3501–3510. doi: 10.1002/j.1460-2075.1989.tb08515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  6. Galli D. M., LeBlanc D. J. Characterization of pVT736-1, a rolling-circle plasmid from the gram-negative bacterium Actinobacillus actinomycetemcomitans. Plasmid. 1994 Mar;31(2):148–157. doi: 10.1006/plas.1994.1016. [DOI] [PubMed] [Google Scholar]
  7. Galli D., Friesenegger A., Wirth R. Transcriptional control of sex-pheromone-inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1-encoded) aggregation substance. Mol Microbiol. 1992 May;6(10):1297–1308. doi: 10.1111/j.1365-2958.1992.tb00851.x. [DOI] [PubMed] [Google Scholar]
  8. Galli D., Wirth R. Comparative analysis of Enterococcus faecalis sex pheromone plasmids identifies a single homologous DNA region which codes for aggregation substance. J Bacteriol. 1991 May;173(9):3029–3033. doi: 10.1128/jb.173.9.3029-3033.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haraszthy V. I., Sunday G. J., Bobek L. A., Motley T. S., Preus H., Zambon J. J. Identification and analysis of the gap region in the 23S ribosomal RNA from Actinobacillus actinomycetemcomitans. J Dent Res. 1992 Sep;71(9):1561–1568. doi: 10.1177/00220345920710090401. [DOI] [PubMed] [Google Scholar]
  10. Hjalt T., Wagner E. G. The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control. Nucleic Acids Res. 1992 Dec 25;20(24):6723–6732. doi: 10.1093/nar/20.24.6723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim K., Meyer R. J. Copy-number of broad host-range plasmid R1162 is regulated by a small RNA. Nucleic Acids Res. 1986 Oct 24;14(20):8027–8046. doi: 10.1093/nar/14.20.8027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kumar C. C., Novick R. P. Plasmid pT181 replication is regulated by two countertranscripts. Proc Natl Acad Sci U S A. 1985 Feb;82(3):638–642. doi: 10.1073/pnas.82.3.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LeBlanc D. J., Lee L. N., Abu-Al-Jaibat A. R., Sreenivasan P. K., Fives-Taylor P. M. Identification of plasmids in Actinobacillus actinomycetemcomitans and construction of intergeneric shuttle plasmids. Oral Microbiol Immunol. 1993 Apr;8(2):94–99. doi: 10.1111/j.1399-302x.1993.tb00552.x. [DOI] [PubMed] [Google Scholar]
  14. Light J., Riise E., Molin S. Transcription and its regulation in the basic replicon region of plasmid R1. Mol Gen Genet. 1985;198(3):503–508. doi: 10.1007/BF00332947. [DOI] [PubMed] [Google Scholar]
  15. Molin S., Stougaard P., Light J., Nordström M., Nordström K. Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control. Mol Gen Genet. 1981;181(1):123–130. doi: 10.1007/BF00339015. [DOI] [PubMed] [Google Scholar]
  16. Novick R. P., Iordanescu S., Projan S. J., Kornblum J., Edelman I. pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator. Cell. 1989 Oct 20;59(2):395–404. doi: 10.1016/0092-8674(89)90300-0. [DOI] [PubMed] [Google Scholar]
  17. Novick R. P. Staphylococcal plasmids and their replication. Annu Rev Microbiol. 1989;43:537–565. doi: 10.1146/annurev.mi.43.100189.002541. [DOI] [PubMed] [Google Scholar]
  18. Olsvik B., Preus H. R. Plasmids in Actinobacillus actinomycetemcomitans strains isolated from periodontal lesions of patients with rapidly destructive periodontitis. Oral Microbiol Immunol. 1989 Dec;4(4):219–221. doi: 10.1111/j.1399-302x.1989.tb00255.x. [DOI] [PubMed] [Google Scholar]
  19. Raab R., Neal G., Sohaskey C., Smith J., Young R. Dominance in lambda S mutations and evidence for translational control. J Mol Biol. 1988 Jan 5;199(1):95–105. doi: 10.1016/0022-2836(88)90381-6. [DOI] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Seery L. T., Nolan N. C., Sharp P. M., Devine K. M. Comparative analysis of the pC194 group of rolling circle plasmids. Plasmid. 1993 Nov;30(3):185–196. doi: 10.1006/plas.1993.1051. [DOI] [PubMed] [Google Scholar]
  22. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  23. Smith R. A., Parkinson J. S. Overlapping genes at the cheA locus of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5370–5374. doi: 10.1073/pnas.77.9.5370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Sreenivasan P. K., LeBlanc D. J., Lee L. N., Fives-Taylor P. Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids. Infect Immun. 1991 Dec;59(12):4621–4627. doi: 10.1128/iai.59.12.4621-4627.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wagner E. G., Simons R. W. Antisense RNA control in bacteria, phages, and plasmids. Annu Rev Microbiol. 1994;48:713–742. doi: 10.1146/annurev.mi.48.100194.003433. [DOI] [PubMed] [Google Scholar]
  27. Zambon J. J. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol. 1985 Jan;12(1):1–20. doi: 10.1111/j.1600-051x.1985.tb01348.x. [DOI] [PubMed] [Google Scholar]
  28. de Smit M. H., van Duin J. Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7668–7672. doi: 10.1073/pnas.87.19.7668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. del Solar G. H., Pérez-Martín J., Espinosa M. Plasmid pLS1-encoded RepA protein regulates transcription from repAB promoter by binding to a DNA sequence containing a 13-base pair symmetric element. J Biol Chem. 1990 Jul 25;265(21):12569–12575. [PubMed] [Google Scholar]
  30. del Solar G. H., de al Campa A. G., Pérez-Martín J., Choli T., Espinosa M. Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLS1. Nucleic Acids Res. 1989 Apr 11;17(7):2405–2420. doi: 10.1093/nar/17.7.2405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. del Solar G., Espinosa M. The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA. Mol Microbiol. 1992 Jan;6(1):83–94. doi: 10.1111/j.1365-2958.1992.tb00840.x. [DOI] [PubMed] [Google Scholar]
  32. del Solar G., Moscoso M., Espinosa M. Rolling circle-replicating plasmids from gram-positive and gram-negative bacteria: a wall falls. Mol Microbiol. 1993 May;8(5):789–796. doi: 10.1111/j.1365-2958.1993.tb01625.x. [DOI] [PubMed] [Google Scholar]
  33. van Biesen T., Söderbom F., Wagner E. G., Frost L. S. Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid. Mol Microbiol. 1993 Oct;10(1):35–43. doi: 10.1111/j.1365-2958.1993.tb00901.x. [DOI] [PubMed] [Google Scholar]

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