Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Jun;174(11):3659–3666. doi: 10.1128/jb.174.11.3659-3666.1992

The Streptomyces glaucescens TcmR protein represses transcription of the divergently oriented tcmR and tcmA genes by binding to an intergenic operator region.

P G Guilfoile 1, C R Hutchinson 1
PMCID: PMC206055  PMID: 1592820

Abstract

Preliminary evidence has been presented by Guilfoile and Hutchinson (J. Bacteriol. 174:3651-3658, 1992) suggesting that the Streptomyces glaucescens TcmR protein is a transcriptional repressor. Here, we extend that work by showing that transcription of the S. glaucescens tcmA gene is inducible by tetracenomycin C and that inactivation of the tcmR gene results in constitutive transcription of the tcmA gene. Gel retardation studies show that the TcmR protein binds to the tcmA-tcmR intergenic region in vitro and that this binding is inhibited by tetracenomycin C. Footprinting experiments demonstrate that the TcmR protein binds to an operator region that encompasses both the tcmA and the tcmR promoters. This genetic and biochemical evidence strongly supports the model of the TcmR protein acting as a repressor in inhibiting transcription of both the tcmA and the tcmR genes, in much the same way that TetR from Tn10 inhibits transcription of tetA and tetR.

Full text

PDF
3659

Images in this article

Selected References

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

  1. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  2. Beck C. F., Mutzel R., Barbé J., Müller W. A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance. J Bacteriol. 1982 May;150(2):633–642. doi: 10.1128/jb.150.2.633-642.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Caballero J. L., Malpartida F., Hopwood D. A. Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture. Mol Gen Genet. 1991 Sep;228(3):372–380. doi: 10.1007/BF00260629. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dhillon N., Hale R. S., Cortes J., Leadlay P. F. Molecular characterization of a gene from Saccharopolyspora erythraea (Streptomyces erythraeus) which is involved in erythromycin biosynthesis. Mol Microbiol. 1989 Oct;3(10):1405–1414. doi: 10.1111/j.1365-2958.1989.tb00123.x. [DOI] [PubMed] [Google Scholar]
  8. Eckert B., Beck C. F. Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential. J Bacteriol. 1989 Jun;171(6):3557–3559. doi: 10.1128/jb.171.6.3557-3559.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fernández-Moreno M. A., Caballero J. L., Hopwood D. A., Malpartida F. The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. Cell. 1991 Aug 23;66(4):769–780. doi: 10.1016/0092-8674(91)90120-n. [DOI] [PubMed] [Google Scholar]
  10. Guilfoile P. G., Hutchinson C. R. A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8553–8557. doi: 10.1073/pnas.88.19.8553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guilfoile P. G., Hutchinson C. R. Sequence and transcriptional analysis of the Streptomyces glaucescens tcmAR tetracenomycin C resistance and repressor gene loci. J Bacteriol. 1992 Jun;174(11):3651–3658. doi: 10.1128/jb.174.11.3651-3658.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hillen W., Gatz C., Altschmied L., Schollmeier K., Meier I. Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions. J Mol Biol. 1983 Sep 25;169(3):707–721. doi: 10.1016/s0022-2836(83)80166-1. [DOI] [PubMed] [Google Scholar]
  13. Hillen W., Schollmeier K., Gatz C. Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region. J Mol Biol. 1984 Jan 15;172(2):185–201. doi: 10.1016/s0022-2836(84)80037-6. [DOI] [PubMed] [Google Scholar]
  14. Kamimiya S., Weisblum B. Translational attenuation control of ermSF, an inducible resistance determinant encoding rRNA N-methyltransferase from Streptomyces fradiae. J Bacteriol. 1988 Apr;170(4):1800–1811. doi: 10.1128/jb.170.4.1800-1811.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kleinschmidt C., Tovar K., Hillen W., Porschke D. Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control. Biochemistry. 1988 Feb 23;27(4):1094–1104. doi: 10.1021/bi00404a003. [DOI] [PubMed] [Google Scholar]
  16. Klock G., Unger B., Gatz C., Hillen W., Altenbuchner J., Schmid K., Schmitt R. Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants. J Bacteriol. 1985 Jan;161(1):326–332. doi: 10.1128/jb.161.1.326-332.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lanzer M., Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973–8977. doi: 10.1073/pnas.85.23.8973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li Y., Dosch D. C., Strohl W. R., Floss H. G. Nucleotide sequence and transcriptional analysis of the nosiheptide-resistance gene from Streptomyces actuosus. Gene. 1990 Jul 2;91(1):9–17. doi: 10.1016/0378-1119(90)90156-l. [DOI] [PubMed] [Google Scholar]
  20. Martín M. F., Liras P. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol. 1989;43:173–206. doi: 10.1146/annurev.mi.43.100189.001133. [DOI] [PubMed] [Google Scholar]
  21. Meier I., Wray L. V., Hillen W. Differential regulation of the Tn10-encoded tetracycline resistance genes tetA and tetR by the tandem tet operators O1 and O2. EMBO J. 1988 Feb;7(2):567–572. doi: 10.1002/j.1460-2075.1988.tb02846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Motamedi H., Hutchinson C. R. Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of Streptomyces glaucescens. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4445–4449. doi: 10.1073/pnas.84.13.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Motamedi H., Wendt-Pienkowski E., Hutchinson C. R. Isolation of tetracenomycin C-nonproducing Streptomyces glaucescens mutants. J Bacteriol. 1986 Aug;167(2):575–580. doi: 10.1128/jb.167.2.575-580.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Postle K., Nguyen T. T., Bertrand K. P. Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant. Nucleic Acids Res. 1984 Jun 25;12(12):4849–4863. doi: 10.1093/nar/12.12.4849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sharrocks A. D., Hornby D. P. S1 nuclease transcript mapping using sequenase-derived single-stranded probes. Biotechniques. 1991 Apr;10(4):426–428. [PubMed] [Google Scholar]
  26. Vara J., Lewandowska-Skarbek M., Wang Y. G., Donadio S., Hutchinson C. R. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J Bacteriol. 1989 Nov;171(11):5872–5881. doi: 10.1128/jb.171.11.5872-5881.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weber W., Zähner H., Siebers J., Schröder K., Zeeck A. Stoffwechselprodukte von Mikroorganismen. 175. Mitteilung. Tetracenomycin C. Arch Microbiol. 1979 May;121(2):111–116. doi: 10.1007/BF00689973. [DOI] [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. Yue S., Motamedi H., Wendt-Pienkowski E., Hutchinson C. R. Anthracycline metabolites of tetracenomycin C-nonproducing Streptomyces glaucescens mutants. J Bacteriol. 1986 Aug;167(2):581–586. doi: 10.1128/jb.167.2.581-586.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES