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. 1993 Jul;175(14):4466–4474. doi: 10.1128/jb.175.14.4466-4474.1993

Repression of the traM gene of plasmid R100 by its own product and integration host factor at one of the two promoters.

T Abo 1, E Ohtsubo 1
PMCID: PMC204887  PMID: 8331074

Abstract

Plasmid R100 codes for the traM gene, which is required for DNA transfer and whose product has been shown to bind to the four sites, called sbmA to sbmD, upstream of traM. To determine whether the TraM protein regulates the expression of traM, we constructed the plasmids carrying various portions of the region upstream of the initiation codon ATG for traM, which was fused with lacZ in frame, and introduced them into the cells, which did or did not harbor another compatible plasmid carrying traM. We then assayed the beta-galactosidase (LacZ) activity to monitor the expression of the fusion genes and analyzed the traM-specific transcripts made in the cells. Two promoters for traM were identified and designated pM1 and pM2. Promoter pM2 lies upstream of pM1 and overlaps the sbmC-sbmD region. Promoter pM1 is constitutively expressed, while pM2 is much stronger but is repressed almost completely by the TraM protein and partially by integration host factor, whose binding site is near pM2. The traM gene is likely to be expressed from pM2 when the TraM protein is at low levels after dilution in the donor cell during cell growth or before its expression in the recipient cell which has just received R100 by conjugation. The expression from pM2 could maintain the amount of the TraM protein at a constant level needed to initiate DNA transfer at any time. Integration host factor, which can partially repress the traM gene, may play a role in forming an active complex with the TraM protein at the sbm region to facilitate DNA transfer.

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Selected References

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  1. Abo T., Inamoto S., Ohtsubo E. Specific DNA binding of the TraM protein to the oriT region of plasmid R100. J Bacteriol. 1991 Oct;173(20):6347–6354. doi: 10.1128/jb.173.20.6347-6354.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Achtman M., Manning P. A., Edelbluth C., Herrlich P. Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4837–4841. doi: 10.1073/pnas.76.10.4837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Armstrong K. A., Acosta R., Ledner E., Machida Y., Pancotto M., McCormick M., Ohtsubo H., Ohtsubo E. A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J Mol Biol. 1984 May 25;175(3):331–348. doi: 10.1016/0022-2836(84)90352-8. [DOI] [PubMed] [Google Scholar]
  5. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  6. Dempsey W. B., Fee B. E. Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100. Mol Microbiol. 1990 Jun;4(6):1019–1028. doi: 10.1111/j.1365-2958.1990.tb00674.x. [DOI] [PubMed] [Google Scholar]
  7. Dempsey W. B. Integration host factor and conjugative transfer of the antibiotic resistance plasmid R100. J Bacteriol. 1987 Sep;169(9):4391–4392. doi: 10.1128/jb.169.9.4391-4392.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dempsey W. B. Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100. Mol Microbiol. 1989 Apr;3(4):561–570. doi: 10.1111/j.1365-2958.1989.tb00202.x. [DOI] [PubMed] [Google Scholar]
  9. Di Laurenzio L., Frost L. S., Paranchych W. The TraM protein of the conjugative plasmid F binds to the origin of transfer of the F and ColE1 plasmids. Mol Microbiol. 1992 Oct;6(20):2951–2959. doi: 10.1111/j.1365-2958.1992.tb01754.x. [DOI] [PubMed] [Google Scholar]
  10. Friedman D. I. Integration host factor: a protein for all reasons. Cell. 1988 Nov 18;55(4):545–554. doi: 10.1016/0092-8674(88)90213-9. [DOI] [PubMed] [Google Scholar]
  11. Gaffney D., Skurray R., Willetts N. Regulation of the F conjugation genes studied by hybridization and tra-lacZ fusion. J Mol Biol. 1983 Jul 25;168(1):103–122. doi: 10.1016/s0022-2836(83)80325-8. [DOI] [PubMed] [Google Scholar]
  12. Gamas P., Caro L., Galas D., Chandler M. Expression of F transfer functions depends on the Escherichia coli integration host factor. Mol Gen Genet. 1987 May;207(2-3):302–305. doi: 10.1007/BF00331593. [DOI] [PubMed] [Google Scholar]
  13. Inamoto S., Ohtsubo E. Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100. J Biol Chem. 1990 Apr 15;265(11):6461–6466. [PubMed] [Google Scholar]
  14. Inamoto S., Yoshioka Y., Ohtsubo E. Identification and characterization of the products from the traJ and traY genes of plasmid R100. J Bacteriol. 1988 Jun;170(6):2749–2757. doi: 10.1128/jb.170.6.2749-2757.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Inamoto S., Yoshioka Y., Ohtsubo E. Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100. J Biol Chem. 1991 Jun 5;266(16):10086–10092. [PubMed] [Google Scholar]
  16. Ippen-Ihler K. A., Minkley E. G., Jr The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet. 1986;20:593–624. doi: 10.1146/annurev.ge.20.120186.003113. [DOI] [PubMed] [Google Scholar]
  17. Kano Y., Ogawa T., Ogura T., Hiraga S., Okazaki T., Imamoto F. Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli. Gene. 1991 Jul 15;103(1):25–30. doi: 10.1016/0378-1119(91)90386-p. [DOI] [PubMed] [Google Scholar]
  18. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matson S. W., Morton B. S. Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT. J Biol Chem. 1991 Aug 25;266(24):16232–16237. [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Mullineaux P., Willetts N. Promoters in the transfer region of plasmid F. Basic Life Sci. 1985;30:605–614. doi: 10.1007/978-1-4613-2447-8_42. [DOI] [PubMed] [Google Scholar]
  22. Reygers U., Wessel R., Müller H., Hoffmann-Berling H. Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor. EMBO J. 1991 Sep;10(9):2689–2694. doi: 10.1002/j.1460-2075.1991.tb07812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Schmid M. B. More than just "histone-like" proteins. Cell. 1990 Nov 2;63(3):451–453. doi: 10.1016/0092-8674(90)90438-k. [DOI] [PubMed] [Google Scholar]
  25. Sharp P. A., Cohen S. N., Davidson N. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. II. Structure of drug resistance (R) factors and F factors. J Mol Biol. 1973 Apr 5;75(2):235–255. doi: 10.1016/0022-2836(73)90018-1. [DOI] [PubMed] [Google Scholar]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. Willetts N., Wilkins B. Processing of plasmid DNA during bacterial conjugation. Microbiol Rev. 1984 Mar;48(1):24–41. doi: 10.1128/mr.48.1.24-41.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Yoshioka Y., Ohtsubo H., Ohtsubo E. Repressor gene finO in plasmids R100 and F: constitutive transfer of plasmid F is caused by insertion of IS3 into F finO. J Bacteriol. 1987 Feb;169(2):619–623. doi: 10.1128/jb.169.2.619-623.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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