Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Dec;175(24):7990–7999. doi: 10.1128/jb.175.24.7990-7999.1993

Expression and purification of the cynR regulatory gene product: CynR is a DNA-binding protein.

A F Lamblin 1, J A Fuchs 1
PMCID: PMC206979  PMID: 8253686

Abstract

The CynR protein, a member of the LysR family, positively regulates the Escherichia coli cyn operon and negatively autoregulates its own transcription. By S1 mapping analysis, the in vivo cynR transcription start site was located 63 bp upstream of the cynTSX operon transcription start site. Topologically, the cynR and cynTSX promoters overlap and direct transcription in opposite directions. The CynR translation initiation codon was identified by oligonucleotide-directed mutagenesis, and the CynR coding sequence was cloned under the control of a T7 phage promoter. The CynR protein was stably expressed at a high level with a T7 RNA polymerase-T7 phage promoter system. Purification by ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation yielded pure CynR protein. Gel shift assays confirmed that CynR is a DNA-binding protein like the other members of the LysR family. The CynR regulatory protein binds specifically to a 136-bp DNA fragment encompassing both the cynR and the cynTSX promoters.

Full text

PDF
7990

Images in this article

7992Image
on p.7992
7995Image
on p.7995
7996Image
on p.7996
7997Image
on p.7997

Selected References

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

  1. 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]
  2. Anderson P. M. Purification and properties of the inducible enzyme cyanase. Biochemistry. 1980 Jun 24;19(13):2882–2888. doi: 10.1021/bi00554a010. [DOI] [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. 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]
  5. 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]
  6. Coco W. M., Rothmel R. K., Henikoff S., Chakrabarty A. M. Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida. J Bacteriol. 1993 Jan;175(2):417–427. doi: 10.1128/jb.175.2.417-427.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  9. Graceffa P., Lehrer S. S. Tropomyosin's end-to-end polymerization is irreversibly lost on exposure to urea or cyanate. Biochem Biophys Res Commun. 1986 Jan 14;134(1):64–70. doi: 10.1016/0006-291x(86)90527-9. [DOI] [PubMed] [Google Scholar]
  10. Guilloton M. B., Lamblin A. F., Kozliak E. I., Gerami-Nejad M., Tu C., Silverman D., Anderson P. M., Fuchs J. A. A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli. J Bacteriol. 1993 Mar;175(5):1443–1451. doi: 10.1128/jb.175.5.1443-1451.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Habeeb L. F., Wang L., Winans S. C. Transcription of the octopine catabolism operon of the Agrobacterium tumor-inducing plasmid pTiA6 is activated by a LysR-type regulatory protein. Mol Plant Microbe Interact. 1991 Jul-Aug;4(4):379–385. doi: 10.1094/mpmi-4-379. [DOI] [PubMed] [Google Scholar]
  12. Hagel P., Gerding J. J., Fieggen W., Bloemendal H. Cyanate formation in solutions of urea. I. Calculation of cyanate concentrations at different temperature and pH. Biochim Biophys Acta. 1971 Sep 28;243(3):366–373. doi: 10.1016/0005-2795(71)90003-1. [DOI] [PubMed] [Google Scholar]
  13. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  14. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [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. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Kusano T., Sugawara K. Specific binding of Thiobacillus ferrooxidans RbcR to the intergenic sequence between the rbc operon and the rbcR gene. J Bacteriol. 1993 Feb;175(4):1019–1025. doi: 10.1128/jb.175.4.1019-1025.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LURIA S. E., BURROUS J. W. Hybridization between Escherichia coli and Shigella. J Bacteriol. 1957 Oct;74(4):461–476. doi: 10.1128/jb.74.4.461-476.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lindquist S., Lindberg F., Normark S. Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC beta-lactamase gene. J Bacteriol. 1989 Jul;171(7):3746–3753. doi: 10.1128/jb.171.7.3746-3753.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ostrowski J., Jagura-Burdzy G., Kredich N. M. DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli. J Biol Chem. 1987 May 5;262(13):5999–6005. [PubMed] [Google Scholar]
  23. Parsek M. R., Shinabarger D. L., Rothmel R. K., Chakrabarty A. M. Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida. J Bacteriol. 1992 Dec;174(23):7798–7806. doi: 10.1128/jb.174.23.7798-7806.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Plamann L. S., Stauffer G. V. Nucleotide sequence of the Salmonella typhimurium metR gene and the metR-metE control region. J Bacteriol. 1987 Sep;169(9):3932–3937. doi: 10.1128/jb.169.9.3932-3937.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reddy P., Peterkofsky A., McKenney K. Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5656–5660. doi: 10.1073/pnas.82.17.5656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ringquist S., Shinedling S., Barrick D., Green L., Binkley J., Stormo G. D., Gold L. Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol Microbiol. 1992 May;6(9):1219–1229. doi: 10.1111/j.1365-2958.1992.tb01561.x. [DOI] [PubMed] [Google Scholar]
  27. Rothmel R. K., Aldrich T. L., Houghton J. E., Coco W. M., Ornston L. N., Chakrabarty A. M. Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family. J Bacteriol. 1990 Feb;172(2):922–931. doi: 10.1128/jb.172.2.922-931.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Schell M. A., Poser E. F. Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. J Bacteriol. 1989 Feb;171(2):837–846. doi: 10.1128/jb.171.2.837-846.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schell M. A., Wender P. E. Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. J Bacteriol. 1986 Apr;166(1):9–14. doi: 10.1128/jb.166.1.9-14.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  33. Stragier P., Patte J. C. Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. III. Nucleotide sequence and regulation of the lysR gene. J Mol Biol. 1983 Aug 5;168(2):333–350. doi: 10.1016/s0022-2836(83)80022-9. [DOI] [PubMed] [Google Scholar]
  34. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  35. Studier F. W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37–44. doi: 10.1016/0022-2836(91)90855-z. [DOI] [PubMed] [Google Scholar]
  36. Sung Y. C., Fuchs J. A. Characterization of the cyn operon in Escherichia coli K12. J Biol Chem. 1988 Oct 15;263(29):14769–14775. [PubMed] [Google Scholar]
  37. Sung Y. C., Fuchs J. A. The Escherichia coli K-12 cyn operon is positively regulated by a member of the lysR family. J Bacteriol. 1992 Jun;174(11):3645–3650. doi: 10.1128/jb.174.11.3645-3650.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sung Y. C., Parsell D., Anderson P. M., Fuchs J. A. Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12. J Bacteriol. 1987 Jun;169(6):2639–2642. doi: 10.1128/jb.169.6.2639-2642.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Urbanowski M. L., Stauffer G. V. Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium. J Bacteriol. 1989 Oct;171(10):5620–5629. doi: 10.1128/jb.171.10.5620-5629.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Urbanowski M. L., Stauffer L. T., Plamann L. S., Stauffer G. V. A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium. J Bacteriol. 1987 Apr;169(4):1391–1397. doi: 10.1128/jb.169.4.1391-1397.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Viale A. M., Kobayashi H., Akazawa T., Henikoff S. rbcR [correction of rcbR], a gene coding for a member of the LysR family of transcriptional regulators, is located upstream of the expressed set of ribulose 1,5-bisphosphate carboxylase/oxygenase genes in the photosynthetic bacterium Chromatium vinosum. J Bacteriol. 1991 Aug;173(16):5224–5229. doi: 10.1128/jb.173.16.5224-5229.1991. [DOI] [PMC free article] [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. 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]
  45. Wek R. C., Hatfield G. W. Nucleotide sequence and in vivo expression of the ilvY and ilvC genes in Escherichia coli K12. Transcription from divergent overlapping promoters. J Biol Chem. 1986 Feb 15;261(5):2441–2450. [PubMed] [Google Scholar]
  46. Wek R. C., Hatfield G. W. Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli. J Mol Biol. 1988 Oct 5;203(3):643–663. doi: 10.1016/0022-2836(88)90199-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES