Control of the ccd operon in plasmid F (original) (raw)

Abstract

The F sex factor plasmid of Escherichia coli contains a pair of genes, ccdA and ccdB, whose protein gene products are involved in an unusual feature of plasmid maintenance. The CcdB protein is a cytotoxin that becomes activated when the F plasmid is lost, thereby killing the F- segregant cells. In F+ cells, the CcdA protein protects against the lethal effects of CcdB. In the present study we show that ccdA and ccdB expressions are negatively autoregulated at the level of transcription. Genetic studies showed that repression required at least ccdB; ccdA alone was without effect, and ccdB alone was not examined because it is lethal. Ccd-operator complexes were purified and contained a mixture of both CcdA and CcdB proteins; however, we could not conclude from our results whether CcdA was necessary for DNA binding or autorepression. By using restriction fragments of the promoter-operator region, we obtained results indicating that at least two DNA-binding sites existed for the Ccd protein(s). Subsequent footprinting of the binding sites showed protection over about a 113-base-pair region encompassing the putative promoter-operator and the beginning of the ccdA gene.

2353

Images in this article

Selected References

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

  1. Bex F., Karoui H., Rokeach L., Drèze P., Garcia L., Couturier M. Mini-F encoded proteins: identification of a new 10.5 kilodalton species. EMBO J. 1983;2(11):1853–1861. doi: 10.1002/j.1460-2075.1983.tb01671.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carey J. Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study. Proc Natl Acad Sci U S A. 1988 Feb;85(4):975–979. doi: 10.1073/pnas.85.4.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hiraga S., Jaffé A., Ogura T., Mori H., Takahashi H. F plasmid ccd mechanism in Escherichia coli. J Bacteriol. 1986 Apr;166(1):100–104. doi: 10.1128/jb.166.1.100-104.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Karoui H., Bex F., Drèze P., Couturier M. Ham22, a mini-F mutation which is lethal to host cell and promotes recA-dependent induction of lambdoid prophage. EMBO J. 1983;2(11):1863–1868. doi: 10.1002/j.1460-2075.1983.tb01672.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kline B. C., Trawick J. Identification and characterization of a second copy number control gene in mini-F plasmids. Mol Gen Genet. 1983;192(3):408–415. doi: 10.1007/BF00392183. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lane D., Hill D., Caughey P., Gunn P. The mini-F primary origin. Sequence analysis and multiple activities. J Mol Biol. 1984 Dec 5;180(2):267–282. doi: 10.1016/s0022-2836(84)80004-2. [DOI] [PubMed] [Google Scholar]
  12. Lane D., de Feyter R., Kennedy M., Phua S. H., Semon D. D protein of miniF plasmid acts as a repressor of transcription and as a site-specific resolvase. Nucleic Acids Res. 1986 Dec 22;14(24):9713–9728. [PMC free article] [PubMed] [Google Scholar]
  13. Levy S. B. Physical and functional characteristics of R-factor deoxyribonucleic acid segregated into Escherichia coli minicells. J Bacteriol. 1971 Oct;108(1):300–308. doi: 10.1128/jb.108.1.300-308.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Linn T., Ralling G. A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ. Plasmid. 1985 Sep;14(2):134–142. doi: 10.1016/0147-619x(85)90073-3. [DOI] [PubMed] [Google Scholar]
  15. Manis J. J., Kline B. C. F plasmid incompatibility and copy number genes: their map locations and interactions. Plasmid. 1978 Sep;1(4):492–507. doi: 10.1016/0147-619x(78)90007-0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Miki T., Chang Z. T., Horiuchi T. Control of cell division by sex factor F in Escherichia coli. II. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment. J Mol Biol. 1984 Apr 25;174(4):627–646. doi: 10.1016/0022-2836(84)90087-1. [DOI] [PubMed] [Google Scholar]
  18. Miki T., Yoshioka K., Horiuchi T. Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA. J Mol Biol. 1984 Apr 25;174(4):605–625. doi: 10.1016/0022-2836(84)90086-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  22. Trawick J. D., Kline B. C. A two-stage molecular model for control of mini-F replication. Plasmid. 1985 Jan;13(1):59–69. doi: 10.1016/0147-619x(85)90056-3. [DOI] [PubMed] [Google Scholar]
  23. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]