Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Mar;177(5):1275–1284. doi: 10.1128/jb.177.5.1275-1284.1995

Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation.

I Kullik 1, M B Toledano 1, L A Tartaglia 1, G Storz 1
PMCID: PMC176734  PMID: 7868602

Abstract

OxyR is a redox-sensitive transcriptional regulator of the LysR family which activates the expression of genes important for the defense against hydrogen peroxide in Escherichia coli and Samonella typhimurium. OxyR is sensitive to oxidation and reduction, and only oxidized OxyR is able to activate transcription of its target genes. Using site-directed mutagenesis, we found that one cysteine residue (C-199) is critical for the redox sensitivity of OxyR, and a C-199-->S mutation appears to lock the OxyR protein in the reduced form. We also used a random mutagenesis approach to isolate eight constitutively active mutants. All of the mutations are located in the C-terminal half of the protein, and four of the mutations map near the critical C-199 residue. In vivo as well as in vitro transcription experiments showed that the constitutive mutant proteins were able to activate transcription under both oxidizing and reducing conditions, and DNase I footprints showed that this activation is due to the ability of the mutant proteins to induce cooperative binding of RNA polymerase. Unexpectedly, RNA polymerase was also found to reciprocally affect OxyR binding.

Full Text

The Full Text of this article is available as a PDF (538.8 KB).

Selected References

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

  1. Adhya S., Garges S. Positive control. J Biol Chem. 1990 Jul 5;265(19):10797–10800. [PubMed] [Google Scholar]
  2. Altuvia S., Almirón M., Huisman G., Kolter R., Storz G. The dps promoter is activated by OxyR during growth and by IHF and sigma S in stationary phase. Mol Microbiol. 1994 Jul;13(2):265–272. doi: 10.1111/j.1365-2958.1994.tb00421.x. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  4. Brosius J., Holy A. Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6929–6933. doi: 10.1073/pnas.81.22.6929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burn J. E., Hamilton W. D., Wootton J. C., Johnston A. W. Single and multiple mutations affecting properties of the regulatory gene nodD of Rhizobium. Mol Microbiol. 1989 Nov;3(11):1567–1577. doi: 10.1111/j.1365-2958.1989.tb00142.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Calos M. P. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature. 1978 Aug 24;274(5673):762–765. doi: 10.1038/274762a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chang M., Crawford I. P. The roles of indoleglycerol phosphate and the TrpI protein in the expression of trpBA from Pseudomonas aeruginosa. Nucleic Acids Res. 1990 Feb 25;18(4):979–988. doi: 10.1093/nar/18.4.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Christman M. F., Morgan R. W., Jacobson F. S., Ames B. N. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell. 1985 Jul;41(3):753–762. doi: 10.1016/s0092-8674(85)80056-8. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Fisher R. F., Long S. R. Rhizobium--plant signal exchange. Nature. 1992 Jun 25;357(6380):655–660. doi: 10.1038/357655a0. [DOI] [PubMed] [Google Scholar]
  13. Hidalgo E., Demple B. An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein. EMBO J. 1994 Jan 1;13(1):138–146. doi: 10.1002/j.1460-2075.1994.tb06243.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huang J. Z., Schell M. A. In vivo interactions of the NahR transcriptional activator with its target sequences. Inducer-mediated changes resulting in transcription activation. J Biol Chem. 1991 Jun 15;266(17):10830–10838. [PubMed] [Google Scholar]
  15. Kamphuis I. G., Kalk K. H., Swarte M. B., Drenth J. Structure of papain refined at 1.65 A resolution. J Mol Biol. 1984 Oct 25;179(2):233–256. doi: 10.1016/0022-2836(84)90467-4. [DOI] [PubMed] [Google Scholar]
  16. 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]
  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. Lin W. S., Armstrong D. A., Gaucher G. M. Formation and repair of papain sulfenic acid. Can J Biochem. 1975 Mar;53(3):298–307. doi: 10.1139/o75-042. [DOI] [PubMed] [Google Scholar]
  19. Makino K., Amemura M., Kim S. K., Nakata A., Shinagawa H. Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli. Genes Dev. 1993 Jan;7(1):149–160. doi: 10.1101/gad.7.1.149. [DOI] [PubMed] [Google Scholar]
  20. McIver J., Djordjevic M. A., Weinman J. J., Bender G. L., Rolfe B. G. Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules. Mol Plant Microbe Interact. 1989 May-Jun;2(3):97–106. doi: 10.1094/mpmi-2-097. [DOI] [PubMed] [Google Scholar]
  21. Ostrowski J., Kredich N. M. Molecular characterization of the cysJIH promoters of Salmonella typhimurium and Escherichia coli: regulation by cysB protein and N-acetyl-L-serine. J Bacteriol. 1989 Jan;171(1):130–140. doi: 10.1128/jb.171.1.130-140.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Poole L. B., Claiborne A. The non-flavin redox center of the streptococcal NADH peroxidase. II. Evidence for a stabilized cysteine-sulfenic acid. J Biol Chem. 1989 Jul 25;264(21):12330–12338. [PubMed] [Google Scholar]
  23. Schell M. A., Brown P. H., Raju S. Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator. J Biol Chem. 1990 Mar 5;265(7):3844–3850. [PubMed] [Google Scholar]
  24. Schell M. A. Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol. 1993;47:597–626. doi: 10.1146/annurev.mi.47.100193.003121. [DOI] [PubMed] [Google Scholar]
  25. Stehle T., Ahmed S. A., Claiborne A., Schulz G. E. Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution. J Mol Biol. 1991 Oct 20;221(4):1325–1344. [PubMed] [Google Scholar]
  26. Storz G., Altuvia S. OxyR regulon. Methods Enzymol. 1994;234:217–223. doi: 10.1016/0076-6879(94)34088-9. [DOI] [PubMed] [Google Scholar]
  27. Storz G., Christman M. F., Sies H., Ames B. N. Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8917–8921. doi: 10.1073/pnas.84.24.8917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Tao K., Fujita N., Ishihama A. Involvement of the RNA polymerase alpha subunit C-terminal region in co-operative interaction and transcriptional activation with OxyR protein. Mol Microbiol. 1993 Mar;7(6):859–864. doi: 10.1111/j.1365-2958.1993.tb01176.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Tao K., Makino K., Yonei S., Nakata A., Shinagawa H. Purification and characterization of the Escherichia coli OxyR protein, the positive regulator for a hydrogen peroxide-inducible regulon. J Biochem. 1991 Feb;109(2):262–266. [PubMed] [Google Scholar]
  32. 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]
  33. Thomas M. S., Glass R. E. Escherichia coli rpoA mutation which impairs transcription of positively regulated systems. Mol Microbiol. 1991 Nov;5(11):2719–2725. doi: 10.1111/j.1365-2958.1991.tb01980.x. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Wang L., Helmann J. D., Winans S. C. The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite. Cell. 1992 May 15;69(4):659–667. doi: 10.1016/0092-8674(92)90229-6. [DOI] [PubMed] [Google Scholar]
  36. Warne S. R., Varley J. M., Boulnois G. J., Norton M. G. Identification and characterization of a gene that controls colony morphology and auto-aggregation in Escherichia coli K12. J Gen Microbiol. 1990 Mar;136(3):455–462. doi: 10.1099/00221287-136-3-455. [DOI] [PubMed] [Google Scholar]

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

RESOURCES