Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Oct 15;24(20):4042–4049. doi: 10.1093/nar/24.20.4042

Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C.

W Sun 1, S Hattman 1
PMCID: PMC146201  PMID: 8918810

Abstract

Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.

Full Text

The Full Text of this article is available as a PDF (154.1 KB).

Selected References

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

  1. Balke V., Nagaraja V., Gindlesperger T., Hattman S. Functionally distinct RNA polymerase binding sites in the phage Mu mom promoter region. Nucleic Acids Res. 1992 Jun 11;20(11):2777–2784. doi: 10.1093/nar/20.11.2777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bukhari A. I., Ambrosio L. The invertible segment of bacteriophage Mu DNA determines the adsorption properties of Mu particles. Nature. 1978 Feb 9;271(5645):575–577. doi: 10.1038/271575a0. [DOI] [PubMed] [Google Scholar]
  3. Bölker M., Kahmann R. The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter. EMBO J. 1989 Aug;8(8):2403–2410. doi: 10.1002/j.1460-2075.1989.tb08370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bölker M., Wulczyn F. G., Kahmann R. Role of bacteriophage Mu C protein in activation of the mom gene promoter. J Bacteriol. 1989 Apr;171(4):2019–2027. doi: 10.1128/jb.171.4.2019-2027.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christman M. F., Storz G., Ames B. N. OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins. Proc Natl Acad Sci U S A. 1989 May;86(10):3484–3488. doi: 10.1073/pnas.86.10.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Gindlesperger T. L., Hattman S. In vitro transcriptional activation of the phage Mu mom promoter by C protein. J Bacteriol. 1994 May;176(10):2885–2891. doi: 10.1128/jb.176.10.2885-2891.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hattman S. DNA methyltransferase-dependent transcription of the phage Mu mom gene. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5518–5521. doi: 10.1073/pnas.79.18.5518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hattman S., Goradia M., Monaghan C., Bukhari A. I. Regulation of the DNA-modification function of bacteriophage Mu. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):647–653. doi: 10.1101/sqb.1983.047.01.076. [DOI] [PubMed] [Google Scholar]
  11. Hattman S., Ives J., Margolin W., Howe M. M. Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region. Gene. 1985;39(1):71–76. doi: 10.1016/0378-1119(85)90109-x. [DOI] [PubMed] [Google Scholar]
  12. Hattman S., Ives J. S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression. Gene. 1984 Jul-Aug;29(1-2):185–198. doi: 10.1016/0378-1119(84)90179-3. [DOI] [PubMed] [Google Scholar]
  13. Hattman S., Newman L., Murthy H. M., Nagaraja V. Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10027–10031. doi: 10.1073/pnas.88.22.10027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kahmann R. Methylation regulates the expression of a DNA-modification function encoded by bacteriophage Mu. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):639–646. doi: 10.1101/sqb.1983.047.01.075. [DOI] [PubMed] [Google Scholar]
  15. Kahmann R., Seiler A., Wulczyn F. G., Pfaff E. The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function. Gene. 1985;39(1):61–70. doi: 10.1016/0378-1119(85)90108-8. [DOI] [PubMed] [Google Scholar]
  16. Kahmann R. The mom gene of bacteriophage Mu. Curr Top Microbiol Immunol. 1984;108:29–47. doi: 10.1007/978-3-642-69370-0_4. [DOI] [PubMed] [Google Scholar]
  17. Kamp D., Kahmann R., Zipser D., Broker T. R., Chow L. T. Inversion of the G DNA segment of phage Mu controls phage infectivity. Nature. 1978 Feb 9;271(5645):577–580. doi: 10.1038/271577a0. [DOI] [PubMed] [Google Scholar]
  18. Keilty S., Rosenberg M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem. 1987 May 5;262(13):6389–6395. [PubMed] [Google Scholar]
  19. Kullik I., Stevens J., Toledano M. B., Storz G. Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for DNA binding and multimerization. J Bacteriol. 1995 Mar;177(5):1285–1291. doi: 10.1128/jb.177.5.1285-1291.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kullik I., Toledano M. B., Tartaglia L. A., Storz G. Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation. J Bacteriol. 1995 Mar;177(5):1275–1284. doi: 10.1128/jb.177.5.1275-1284.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Margolin W., Rao G., Howe M. M. Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence. J Bacteriol. 1989 Apr;171(4):2003–2018. doi: 10.1128/jb.171.4.2003-2018.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marinus M. G., Carraway M., Frey A. Z., Brown L., Arraj J. A. Insertion mutations in the dam gene of Escherichia coli K-12. Mol Gen Genet. 1983;192(1-2):288–289. doi: 10.1007/BF00327681. [DOI] [PubMed] [Google Scholar]
  23. Nair T. M., Kulkarni B. D., Nagaraja V. Differential binding of RNA polymerase to the wild type Mu mom promoter and its C independent mutant: a theoretical analysis. Biophys Chem. 1995 Feb;53(3):241–245. doi: 10.1016/0301-4622(94)00116-2. [DOI] [PubMed] [Google Scholar]
  24. Plasterk R. H., Vrieling H., Van de Putte P. Transcription initiation of Mu mom depends on methylation of the promoter region and a phage-coded transactivator. Nature. 1983 Jan 27;301(5898):344–347. doi: 10.1038/301344a0. [DOI] [PubMed] [Google Scholar]
  25. Sasse-Dwight S., Gralla J. D. Probing co-operative DNA-binding in vivo. The lac O1:O3 interaction. J Mol Biol. 1988 Jul 5;202(1):107–119. doi: 10.1016/0022-2836(88)90523-2. [DOI] [PubMed] [Google Scholar]
  26. Storz G., Tartaglia L. A., Ames B. N. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science. 1990 Apr 13;248(4952):189–194. doi: 10.1126/science.2183352. [DOI] [PubMed] [Google Scholar]
  27. Swinton D., Hattman S., Crain P. F., Cheng C. S., Smith D. L., McCloskey J. A. Purification and characterization of the unusual deoxynucleoside, alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage Mu modification function. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7400–7404. doi: 10.1073/pnas.80.24.7400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tao K., Makino K., Yonei S., Nakata A., Shinagawa H. Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins. Mol Gen Genet. 1989 Sep;218(3):371–376. doi: 10.1007/BF00332397. [DOI] [PubMed] [Google Scholar]
  29. Tartaglia L. A., Storz G., Ames B. N. Identification and molecular analysis of oxyR-regulated promoters important for the bacterial adaptation to oxidative stress. J Mol Biol. 1989 Dec 20;210(4):709–719. doi: 10.1016/0022-2836(89)90104-6. [DOI] [PubMed] [Google Scholar]
  30. Toledano M. B., Kullik I., Trinh F., Baird P. T., Schneider T. D., Storz G. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell. 1994 Sep 9;78(5):897–909. doi: 10.1016/s0092-8674(94)90702-1. [DOI] [PubMed] [Google Scholar]
  31. Toussaint A. The DNA modification function of temperate phage Mu-1. Virology. 1976 Mar;70(1):17–27. doi: 10.1016/0042-6822(76)90232-4. [DOI] [PubMed] [Google Scholar]
  32. Wulczyn F. G., Kahmann R. Translational stimulation: RNA sequence and structure requirements for binding of Com protein. Cell. 1991 Apr 19;65(2):259–269. doi: 10.1016/0092-8674(91)90160-z. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES