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. 1989 Aug;8(8):2403–2410. doi: 10.1002/j.1460-2075.1989.tb08370.x

The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter.

M Bölker 1, R Kahmann 1
PMCID: PMC401183  PMID: 2551682

Abstract

Expression of the phage Mu mom gene is transcriptionally regulated by DNA methylation. Three GATC sites upstream of the mom promoter have to be methylated by the Escherichia coli deoxyadenosine methylase (Dam) to allow initiation of transcription. An E. coli dam strain was mutagenized with Tn5 in an attempt to isolate mutants which allow mom gene expression. Three independent Tn5 mutants were isolated, each mapped to a gene at 89.6 min which we designate momR. The wildtype gene was cloned and sequenced, it encodes a protein of 305 amino acids. The protein belongs to a group of related bacterial activators recently identified as the LysR family (Henikoff et al., 1988). MomR protein was overproduced and purified. Expression of momR is autoregulated; MomR binds to a 43 bp region upstream of its coding sequence. In the mom promoter MomR protects a 43 bp region containing the three GATC sites. Specific binding to these sequences was observed only with unmethylated DNA. Fortuitously, we learned that MomR is identical to OxyR, a regulatory protein responding to oxidative stress. We discuss the implications of this control for Mu development.

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Selected References

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  1. Appleyard R K. Segregation of Lambda Lysogenicity during Bacterial Recombination in Escherichia Coli K12. Genetics. 1954 Jul;39(4):429–439. doi: 10.1093/genetics/39.4.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Braun R. E., Wright A. DNA methylation differentially enhances the expression of one of the two E. coli dnaA promoters in vivo and in vitro. Mol Gen Genet. 1986 Feb;202(2):246–250. doi: 10.1007/BF00331644. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Chang M., Hadero A., Crawford I. P. Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes. J Bacteriol. 1989 Jan;171(1):172–183. doi: 10.1128/jb.171.1.172-183.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Dabbs E. R. The ribosomal components responsible for kasugamycin dependence, and its suppression, in a mutant of Escherichia coli. Mol Gen Genet. 1980 Jan;177(2):271–276. doi: 10.1007/BF00267438. [DOI] [PubMed] [Google Scholar]
  10. Devine E. A., Moran M. C., Jederlinic P. J., Mazaitis A. J., Vogel H. J. EcoRI cleavage sites in the argECBH region of the Escherichia coli chromosome. J Bacteriol. 1977 Feb;129(2):1072–1077. doi: 10.1128/jb.129.2.1072-1077.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Heisig P., Kahmann R. The sequence and mom-transactivation function of the C gene of bacteriophage Mu. Gene. 1986;43(1-2):59–67. doi: 10.1016/0378-1119(86)90008-9. [DOI] [PubMed] [Google Scholar]
  15. Henikoff S., Haughn G. W., Calvo J. M., Wallace J. C. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6602–6606. doi: 10.1073/pnas.85.18.6602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hertzberg R. P., Dervan P. B. Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses. Biochemistry. 1984 Aug 14;23(17):3934–3945. doi: 10.1021/bi00312a022. [DOI] [PubMed] [Google Scholar]
  17. Howe M. M. Prophage deletion mapping of bacteriophage Mu-1. Virology. 1973 Jul;54(1):93–101. doi: 10.1016/0042-6822(73)90118-9. [DOI] [PubMed] [Google Scholar]
  18. Jacobson F. S., Morgan R. W., Christman M. F., Ames B. N. An alkyl hydroperoxide reductase from Salmonella typhimurium involved in the defense of DNA against oxidative damage. Purification and properties. J Biol Chem. 1989 Jan 25;264(3):1488–1496. [PubMed] [Google Scholar]
  19. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  24. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  25. Kücherer C., Lother H., Kölling R., Schauzu M. A., Messer W. Regulation of transcription of the chromosomal dnaA gene of Escherichia coli. Mol Gen Genet. 1986 Oct;205(1):115–121. doi: 10.1007/BF02428040. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Marinus M. G. DNA methylation influences trpR promoter activity in Escherichia coli K-12. Mol Gen Genet. 1985;200(1):185–186. doi: 10.1007/BF00383334. [DOI] [PubMed] [Google Scholar]
  28. Marinus M. G., Morris N. R. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol. 1973 Jun;114(3):1143–1150. doi: 10.1128/jb.114.3.1143-1150.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  31. Miller B. E., Kredich N. M. Purification of the cysB protein from Salmonella typhimurium. J Biol Chem. 1987 May 5;262(13):6006–6009. [PubMed] [Google Scholar]
  32. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  33. Peterson K. R., Wertman K. F., Mount D. W., Marinus M. G. Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon. Mol Gen Genet. 1985;201(1):14–19. doi: 10.1007/BF00397979. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Plumbridge J., Söll D. The effect of dam methylation on the expression of glnS in E. coli. Biochimie. 1987 May;69(5):539–541. doi: 10.1016/0300-9084(87)90091-5. [DOI] [PubMed] [Google Scholar]
  36. Roberts D., Hoopes B. C., McClure W. R., Kleckner N. IS10 transposition is regulated by DNA adenine methylation. Cell. 1985 Nov;43(1):117–130. doi: 10.1016/0092-8674(85)90017-0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Schauder B., Blöcker H., Frank R., McCarthy J. E. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene. 1987;52(2-3):279–283. doi: 10.1016/0378-1119(87)90054-0. [DOI] [PubMed] [Google Scholar]
  39. Schauzu M. A., Kücherer C., Kölling R., Messer W., Lother H. Transcripts within the replication origin, oriC, of Escherichia coli. Nucleic Acids Res. 1987 Mar 25;15(6):2479–2497. doi: 10.1093/nar/15.6.2479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Seiler A., Blöcker H., Frank R., Kahmann R. The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression. EMBO J. 1986 Oct;5(10):2719–2728. doi: 10.1002/j.1460-2075.1986.tb04556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. Wulczyn F. G., Kahmann R. Post-transcriptional regulation of the bacteriophage Mu mom gene by the com gene product. Gene. 1987;51(2-3):139–147. doi: 10.1016/0378-1119(87)90302-7. [DOI] [PubMed] [Google Scholar]

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