Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Jan;3(1):57–63. doi: 10.1002/j.1460-2075.1984.tb01761.x

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

C M Thomas, M A Cross, A A Hussain, C A Smith
PMCID: PMC557297  PMID: 6323170

Abstract

Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative bacterial species. To analyze the elements of its control mechanism, we have measured the copy number in Escherichia coli of mini-RK2 replicons isogenic except for defined deletions in regions adjacent to the vegetative replication origin, oriVRK2, which have previously been implicated in copy number control because of their expression of plasmid incompatibility. The results indicate that while the previously defined 700-bp HaeII oriVRK2 fragment carries one copy control element (copA), a second (copB) lies at least partly outside this fragment towards the tetracycline resistance genes of RK2. Deletions affecting both these regions give a mini replicon with a copy number of 35-40 compared with 4-7 for parental RK2. Further incompatibility experiments indicate that targets for both incA (copA) and incB (copB) lie within the 700-bp HaeII oriVRK2 fragment.

Full text

PDF
57

Images in this article

58Fig. 1.
on p.58

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beringer J. E. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974 Sep;84(1):188–198. doi: 10.1099/00221287-84-1-188. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Brady G., Frey J., Danbara H., Timmis K. N. Replication control mutations of plasmid R6-5 and their effects on interactions of the RNA-I control element with its target. J Bacteriol. 1983 Apr;154(1):429–436. doi: 10.1128/jb.154.1.429-436.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burkardt H. J., Riess G., Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. doi: 10.1099/00221287-114-2-341. [DOI] [PubMed] [Google Scholar]
  6. Cesareni G., Muesing M. A., Polisky B. Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6313–6317. doi: 10.1073/pnas.79.20.6313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cho J. J., Panopoulos N. J., Schroth M. N. Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwinia species. J Bacteriol. 1975 Apr;122(1):192–198. doi: 10.1128/jb.122.1.192-198.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Datta N., Hedges R. W. Host ranges of R factors. J Gen Microbiol. 1972 May;70(3):453–460. doi: 10.1099/00221287-70-3-453. [DOI] [PubMed] [Google Scholar]
  9. Figurski D. H., Meyer R. J., Helinski D. R. Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro. J Mol Biol. 1979 Sep 25;133(3):295–318. doi: 10.1016/0022-2836(79)90395-4. [DOI] [PubMed] [Google Scholar]
  10. Grosschedl R., Hobom G. DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages. Nature. 1979 Feb 22;277(5698):621–627. doi: 10.1038/277621a0. [DOI] [PubMed] [Google Scholar]
  11. Guiney D. G., Helinski D. R. The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer. Mol Gen Genet. 1979 Oct 3;176(2):183–189. doi: 10.1007/BF00273212. [DOI] [PubMed] [Google Scholar]
  12. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  15. Lacatena R. M., Cesareni G. Base pairing of RNA I with its complementary sequence in the primer precursor inhibits ColE1 replication. Nature. 1981 Dec 17;294(5842):623–626. doi: 10.1038/294623a0. [DOI] [PubMed] [Google Scholar]
  16. Light J., Molin S. Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule. EMBO J. 1983;2(1):93–98. doi: 10.1002/j.1460-2075.1983.tb01387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  18. Meyer R. J. Expression of incompatibility by derivatives of the broad host-range inc P-1 plasmid RK2. Mol Gen Genet. 1979;177(1):155–161. doi: 10.1007/BF00267265. [DOI] [PubMed] [Google Scholar]
  19. Meyer R., Figurski D., Helinski D. R. Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2. Mol Gen Genet. 1977 Apr 29;152(3):129–135. doi: 10.1007/BF00268809. [DOI] [PubMed] [Google Scholar]
  20. Meyer R., Hinds M. Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2. J Bacteriol. 1982 Dec;152(3):1078–1090. doi: 10.1128/jb.152.3.1078-1090.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Moore D. D., Denniston-Thompson K., Kruger K. E., Furth M. E., Williams B. G., Daniels D. L., Blattner F. R. Dissection and comparative anatomy of the origins of replication of lambdoid phages. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):155–163. doi: 10.1101/sqb.1979.043.01.022. [DOI] [PubMed] [Google Scholar]
  23. Muesing M., Tamm J., Shepard H. M., Polisky B. A single base-pair alteration is responsible for the DNA overproduction phenotype of a plasmid copy-number mutant. Cell. 1981 Apr;24(1):235–242. doi: 10.1016/0092-8674(81)90519-5. [DOI] [PubMed] [Google Scholar]
  24. Murotsu T., Matsubara K., Sugisaki H., Takanami M. Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene. 1981 Nov;15(2-3):257–271. doi: 10.1016/0378-1119(81)90135-9. [DOI] [PubMed] [Google Scholar]
  25. Oka A., Nomura N., Morita M., Sugisaki H., Sugimoto K., Takanami M. Nucleotide sequence of small ColE1 derivatives: structure of the regions essential for autonomous replication and colicin E1 immunity. Mol Gen Genet. 1979 May 4;172(2):151–159. doi: 10.1007/BF00268276. [DOI] [PubMed] [Google Scholar]
  26. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  27. Olsen R. H., Shipley P. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J Bacteriol. 1973 Feb;113(2):772–780. doi: 10.1128/jb.113.2.772-780.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ryder T. B., Davidson D. B., Rosen J. I., Ohtsubo E., Ohtsubo H. Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichia coli. Gene. 1982 Mar;17(3):299–310. doi: 10.1016/0378-1119(82)90146-9. [DOI] [PubMed] [Google Scholar]
  29. Sakanyan V. A., Yakubov L. Z., Alikhanian S. I., Stepanov A. I. Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4--ColE1). Mol Gen Genet. 1978 Oct 24;165(3):331–341. doi: 10.1007/BF00332534. [DOI] [PubMed] [Google Scholar]
  30. Smith C. A., Thomas C. M. Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2. Mol Gen Genet. 1983;190(2):245–254. doi: 10.1007/BF00330647. [DOI] [PubMed] [Google Scholar]
  31. Stalker D. M., Kolter R., Helinski D. R. Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1150–1154. doi: 10.1073/pnas.76.3.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stalker D. M., Thomas C. M., Helinski D. R. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):8–12. doi: 10.1007/BF00338997. [DOI] [PubMed] [Google Scholar]
  33. Stougaard P., Molin S., Nordström K., Hansen F. G. The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19. Mol Gen Genet. 1981;181(1):116–122. doi: 10.1007/BF00339014. [DOI] [PubMed] [Google Scholar]
  34. Stougaard P., Molin S., Nordström K. RNAs involved in copy-number control and incompatibility of plasmid R1. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6008–6012. doi: 10.1073/pnas.78.10.6008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stueber D., Bujard H. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1982;1(11):1399–1404. doi: 10.1002/j.1460-2075.1982.tb01329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thomas C. M. Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1. Plasmid. 1981 May;5(3):277–291. doi: 10.1016/0147-619x(81)90005-6. [DOI] [PubMed] [Google Scholar]
  37. Thomas C. M. Instability of a high-copy-number mutant of a miniplasmid derived from broad host range IncP plasmid RK2. Plasmid. 1983 Sep;10(2):184–195. doi: 10.1016/0147-619x(83)90071-9. [DOI] [PubMed] [Google Scholar]
  38. Thomas C. M., Meyer R., Helinski D. R. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol. 1980 Jan;141(1):213–222. doi: 10.1128/jb.141.1.213-222.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Thomas C. M. Molecular genetics of broad host range plasmid RK2. Plasmid. 1981 Jan;5(1):10–19. doi: 10.1016/0147-619x(81)90074-3. [DOI] [PubMed] [Google Scholar]
  40. Thomas C. M., Stalker D. M., Helinski D. R. Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):1–7. doi: 10.1007/BF00338996. [DOI] [PubMed] [Google Scholar]
  41. Tolun A., Helinski D. R. Direct repeats of the F plasmid incC region express F incompatibility. Cell. 1981 Jun;24(3):687–694. doi: 10.1016/0092-8674(81)90095-7. [DOI] [PubMed] [Google Scholar]
  42. Tomizawa J., Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6096–6100. doi: 10.1073/pnas.78.10.6096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tomizawa J., Itoh T., Selzer G., Som T. Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1421–1425. doi: 10.1073/pnas.78.3.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  45. Waters S. H., Rogowsky P., Grinsted J., Altenbuchner J., Schmitt R. The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis. Nucleic Acids Res. 1983 Sep 10;11(17):6089–6105. doi: 10.1093/nar/11.17.6089. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES