Molecular and General Genetics MGG

, Volume 217, Issue 2–3, pp 481–487 | Cite as

Specificity of the interactions between the rep proteins and the origins of replication ofStaphylococcus aureus plasmids pT181 and pC221

  • Serban Iordanescu


pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.

Key words

Plasmid pT181 Plasmid pC221 Rep proteins Replication origins Cross-complementation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Carleton S, Projan SJ, Highlander SK, Moghazeh SM, Novick RP (1984) Control of pT181 replication. II. Mutational analysis. EMBO J 3: 657–662Google Scholar
  2. Dotto GP, Zinder ND (1984a) Inereased intracellular concentration of an initiator protein markedly reduces the minimal sequence required for initiation of DNA synthesis. Proc Natl Acad Sci USA 81: 1336–1340PubMedCrossRefGoogle Scholar
  3. Dotto GP, Zinder ND (1984b) Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initator protein. Nature 311: 279–280PubMedCrossRefGoogle Scholar
  4. Dotto GP, Horiuchi K, Zinder ND (1984) The functional origin of bacteriophage f1 DNA replication. Its signals and domains. J Mol Biol 172: 507–521PubMedGoogle Scholar
  5. Eisenberg S, Griffith J, Kornberg A (1977) ϕX174 cistron A protein is a multifunctional enzyme in DNA replication. Proc Natl Acad Sci USA 74: 3198–3202PubMedCrossRefGoogle Scholar
  6. Francke B, Ray DS (1972)Cis-limited action of the gene A product of bacteriophage ϕ X174 and the essential bacterial site. Proc Natl Acad Sci USA 69: 475–479PubMedCrossRefGoogle Scholar
  7. Gennaro ML, Iordanescu S, Novick RP, Murray RW, Steck TR, Khan SA (1989) Functional organization of the plasmid pT181 replication origin. J Mol Biol 205: 355–362PubMedCrossRefGoogle Scholar
  8. Gotz FS, Ahrne S, Lindberg M (1981) Plasmid transfer and genetic recombination by protoplast fusion in staphylococci. J Bacteriol 145: 74–81PubMedGoogle Scholar
  9. Gruss AD, Ross HF, Novick RP (1987) Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc Natl Acad Sci USA 84: 2165–2169PubMedCrossRefGoogle Scholar
  10. Horiuchi K (1980) Origin of DNA replication of bacteriophage f1 as the signal for termination. Proc Natl Acad Sci USA 77: 5226–5229PubMedCrossRefGoogle Scholar
  11. Iordanescu S (1983)Staphylococcus aureus chromosomal mutation specifically affecting the copy number of Inc3 plasmids. Plasmid 10: 130–137PubMedCrossRefGoogle Scholar
  12. Iordanescu S (1987) The Inc3B determinant of plasmid pT181. A mutational analysis. Mol Gen Genet 207: 60–67PubMedCrossRefGoogle Scholar
  13. Iordanescu S, Projan SJ (1988) Replication termination for staphylococcal plasmids: Plasmids pT181 and pC221 cross-react in the termination process. J Bacteriol 170: 3427–3434PubMedGoogle Scholar
  14. Iordanescu S, Surdeanu M (1980) Complementation of a plasmid replication defect by autonomous incompatible plasmids inStaphylococcus aureus. Plasmid 4: 1–7PubMedCrossRefGoogle Scholar
  15. Iordanescu S, Surdeanu M, DellaLatta P, Novick R (1978) Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker. Plasmid 1: 468–479PubMedCrossRefGoogle Scholar
  16. Khan SA, Novick RP (1983) Complete nucleotide sequence of pT181, a tetracycline resistance plasmid fromStaphyloccus aureus. Plasmid 10: 251–259PubMedCrossRefGoogle Scholar
  17. Khan SA, Carleton SM, Novick RP (1981) Replication of plasmid pT181 DNAin vitro: Requirement for a plasmid-encoded product. Proc Natl Acad Sci USA 78: 4902–4906PubMedCrossRefGoogle Scholar
  18. Khan SA, Adler GK, Novick RP (1982) Functional origin of replication of pT181 plasmid DNA is contained within a 168-base-pair segment. Proc Natl Acad Sci USA 79: 4580–4584PubMedCrossRefGoogle Scholar
  19. Kim MH, Ray DS (1985) Mutational mechanisms by which an inactive replication origin of bacteriophage M13 is turned on are similar to mechanisms of activation ofras proto-oncogenes. J Virol 53: 871–878PubMedGoogle Scholar
  20. Koepsel RR, Murray RW, Rosenblum WD, Khan SA (1985) The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities. Proc Natl Acad Sci USA 82: 6845–6849PubMedCrossRefGoogle Scholar
  21. Koepsel RR, Murray RW, Khan SA (1986) Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication. Proc Natl Acad Sci USA 83: 5484–5488PubMedCrossRefGoogle Scholar
  22. Kumar CC, Novick RP (1985) Plasmid pT181 replication is regulated by two countertranscripts. Proc Natl Acad Sci USA 82: 638–642PubMedCrossRefGoogle Scholar
  23. Maniatis T, Fritsch EF, Sambrook J (1983) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  24. Masai H, Kaziro Y, Arai K (1983) Definition oforiR, the minimum DNA segment essential for initiation of R1 plasmid replicationin vitro. Proc Natl Acad Sci USA 80: 6814–6818PubMedCrossRefGoogle Scholar
  25. Monod M, DeNoya C, Dubnau D (1986) Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid fromBacillus subtilis. J Bacteriol 167: 138–147PubMedGoogle Scholar
  26. Novick RP, Murphy E, Gryczan TJ, Baron E, Edelman I (1979) Penicillinase plasmids ofStaphylococcus aureaus. Restriction deletion maps. Plasmid 2: 109–129PubMedCrossRefGoogle Scholar
  27. Novick RP, Adler GK, Majumder S, Khan SA, Carleton S, Rosenblum WD, Iordanescu S (1982) Coding sequence for the pT181repC product: A plasmid-coded protein uniquely required for replication. Proc Natl Acad Sci USA 79: 4108–4112PubMedCrossRefGoogle Scholar
  28. Novick RP, Adler GK, Projan SJ, Carleton S, Highlander SK, Gruss A, Khan SA, Iordanescu S (1984) Control of pT181 replication. I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein. EMBO J 3: 2399–2405PubMedGoogle Scholar
  29. Novick RP, Projan SJ, Kumar CC, Carleton S, Gruss A, Highlander SK, Kornblum J (1985) Replication control for pT181, an indirectly regulated plasmid. In: Helinski DR, Cohen SN, Clewell DB, Jackson DA, Hollaender A (eds) Plasmids in bacteria. Plenum, pp 299–320Google Scholar
  30. Oertel W, Kollek R, Beck E, Goebel W (1979) The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd-19. Mol Gen Genet 171: 277–285PubMedCrossRefGoogle Scholar
  31. Projan SJ, Novick R (1988) Comparative analysis of five related staphylococcal plasmids. Plasmid 19: 203–221PubMedCrossRefGoogle Scholar
  32. Projan SJ, Kornblum J, Moghazeh SL, Edelman I, Gennaro ML, Novick RP (1985) Comparative sequence and functional analysis of pT181 and pC221, cognate plasmid replicons fromStaphylococcus aureus. Mol Gen Genet 199: 452–464PubMedCrossRefGoogle Scholar
  33. Sanger F, Niklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467PubMedCrossRefGoogle Scholar
  34. Selzer G, Som T, Itoh T, Tomizawa J (1983) The origin of replication of plasmid p15A and comparative studies of the nucleotide sequences around the origin of related plasmids. Cell 32: 119–129PubMedCrossRefGoogle Scholar
  35. Stalker DM, Kolter R, Helinski DR (1982) Plasmid R6K replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol 161: 33–43PubMedCrossRefGoogle Scholar
  36. Tolun A, Helinski DR (1982) Separation of the minimal replication region of the F plasmid into a replication origin segment and atrans-acting segment. Mol Gen Genet 186: 372–377PubMedCrossRefGoogle Scholar
  37. van der Ende A, Teertstra R, Wisbeek PJ (1982) Initiation and termination of the bacteriophage ϕX174 rolling circle replicationin vivo: packaging of plasmid single-stranded DNA into bacteriophage ϕX174 coats. Nucleic Acids Res 10: 6849–6863PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Serban Iordanescu
    • 1
  1. 1.Department of Plasmid BiologyThe Public Health Research InstituteNew YorkUSA

Personalised recommendations