Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 May;174(9):2881–2890. doi: 10.1128/jb.174.9.2881-2890.1992

Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression.

C S Barbier 1, S A Short 1
PMCID: PMC205940  PMID: 1569019

Abstract

In Escherichia coli, transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. Transcription initiation of cistrons in this gene family is activated by cyclic AMP-catabolite activator protein (cAMP-CAP), repressed by the CytR protein, and induced by cytidine. A random proofreading mutagenesis procedure and a genetic screen using udp-lac fusions have allowed the identification of distinct regions of the 341-amino-acid CytR polypeptide that are critical for repression of gene expression and response to induction. Determination of the ability of various CytR mutants to control gene expression in vivo indicated that the intrinsic affinity of the CytR protein for operator DNA is gene specific and that efficient repression of transcription by wild-type CytR is dependent on the interaction of CytR with cAMP-CAP. CytR mutants that were cytidine induction defective (CID) were characterized; these mutant proteins had only Asp-281 replaced. Data obtained with cytR delta M149, a dominant negative allele, indicated that the native CytR repressor is an oligomeric protein. Representative cytR mutations were combined with cytR delta M149, and the resulting hybrid repressors were tested for transdominance in a CytR+ E. coli strain. Amino acid substitutions A209E and C289Y suppressed the transdominance of CytR delta M149, suggesting that these replacements alter the normal protein contacts involved in repressor subunit-subunit association. In contrast, amino acid substitutions located in the N-terminal portion of the CytR protein had no effect on the transdominance of CytR delta M149. The results from this study suggest that the CytR repressor is an oligomeric, allosteric protein in which conformational changes are required for repression and derepression.

Full text

PDF
2881

Images in this article

2885Image
on p.2885

Selected References

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

  1. Aldea M., Maples V. F., Kushner S. R. Generation of a detailed physical and genetic map of the ilv-metE-udp region of the Escherichia coli chromosome. J Mol Biol. 1988 Apr 5;200(3):427–438. doi: 10.1016/0022-2836(88)90533-5. [DOI] [PubMed] [Google Scholar]
  2. Barbier C. S., Short S. A. Studies on deo operon regulation in Escherichia coli: cloning and expression of the cytR structural gene. Gene. 1985;36(1-2):37–44. doi: 10.1016/0378-1119(85)90067-8. [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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brennan R. G., Matthews B. W. Structural basis of DNA-protein recognition. Trends Biochem Sci. 1989 Jul;14(7):286–290. doi: 10.1016/0968-0004(89)90066-2. [DOI] [PubMed] [Google Scholar]
  5. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  6. Cox E. C., Horner D. L. Dominant mutators in Escherichia coli. Genetics. 1982 Jan;100(1):7–18. doi: 10.1093/genetics/100.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cox E. C., Horner D. L. Structure and coding properties of a dominant Escherichia coli mutator gene, mutD. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2295–2299. doi: 10.1073/pnas.80.8.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gerlach P., Søgaard-Andersen L., Pedersen H., Martinussen J., Valentin-Hansen P., Bremer E. The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12. J Bacteriol. 1991 Sep;173(17):5419–5430. doi: 10.1128/jb.173.17.5419-5430.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gerlach P., Valentin-Hansen P., Bremer E. Transcriptional regulation of the cytR repressor gene of Escherichia coli: autoregulation and positive control by the cAMP/CAP complex. Mol Microbiol. 1990 Mar;4(3):479–488. doi: 10.1111/j.1365-2958.1990.tb00614.x. [DOI] [PubMed] [Google Scholar]
  10. Gordon A. J., Burns P. A., Fix D. F., Yatagai F., Allen F. L., Horsfall M. J., Halliday J. A., Gray J., Bernelot-Moens C., Glickman B. W. Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein. J Mol Biol. 1988 Mar 20;200(2):239–251. doi: 10.1016/0022-2836(88)90237-9. [DOI] [PubMed] [Google Scholar]
  11. Hammer-Jespersen K., Munch-Ptersen A. Multiple regulation of nucleoside catabolizing enzymes: regulation of the deo operon by the cytR and deoR gene products. Mol Gen Genet. 1975;137(4):327–335. doi: 10.1007/BF00703258. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Mironov A. S., Sukhodolets V. V. Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene. J Bacteriol. 1979 Feb;137(2):802–810. doi: 10.1128/jb.137.2.802-810.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Munch-Petersen A., Nygaard P., Hammer-Jespersen K., Fiil N. Mutants constitutive for nucleoside-catabolizing enzymes in Escherichia coli K12. Isolation, charactrization and mapping. Eur J Biochem. 1972 May 23;27(2):208–215. doi: 10.1111/j.1432-1033.1972.tb01828.x. [DOI] [PubMed] [Google Scholar]
  15. O'Farrell P. H., Kutter E., Nakanishi M. A restriction map of the bacteriophage T4 genome. Mol Gen Genet. 1980;179(2):421–435. doi: 10.1007/BF00425473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Pedersen H., Søgaard-Andersen L., Holst B., Valentin-Hansen P. Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter. J Biol Chem. 1991 Sep 25;266(27):17804–17808. [PubMed] [Google Scholar]
  18. Reidl J., Römisch K., Ehrmann M., Boos W. MalI, a novel protein involved in regulation of the maltose system of Escherichia coli, is highly homologous to the repressor proteins GalR, CytR, and LacI. J Bacteriol. 1989 Sep;171(9):4888–4899. doi: 10.1128/jb.171.9.4888-4899.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rolfes R. J., Zalkin H. Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator. J Biol Chem. 1988 Dec 25;263(36):19653–19661. [PubMed] [Google Scholar]
  20. 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]
  21. Short S. A., Singer J. T. Studies on deo operon regulation in Escherichia coli: cloning and expression of the deoR structural gene. Gene. 1984 Nov;31(1-3):205–211. doi: 10.1016/0378-1119(84)90211-7. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Singer J. T., Barbier C. S., Short S. A. Identification of the Escherichia coli deoR and cytR gene products. J Bacteriol. 1985 Sep;163(3):1095–1100. doi: 10.1128/jb.163.3.1095-1100.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Søgaard-Andersen L., Martinussen J., Møllegaard N. E., Douthwaite S. R., Valentin-Hansen P. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12. J Bacteriol. 1990 Oct;172(10):5706–5713. doi: 10.1128/jb.172.10.5706-5713.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Søgaard-Andersen L., Mironov A. S., Pedersen H., Sukhodelets V. V., Valentin-Hansen P. Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4921–4925. doi: 10.1073/pnas.88.11.4921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Søgaard-Andersen L., Pedersen H., Holst B., Valentin-Hansen P. A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon. Mol Microbiol. 1991 Apr;5(4):969–975. doi: 10.1111/j.1365-2958.1991.tb00772.x. [DOI] [PubMed] [Google Scholar]
  27. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  28. Valentin-Hansen P., Aiba H., Schümperli D. The structure of tandem regulatory regions in the deo operon of Escherichia coli K12. EMBO J. 1982;1(3):317–322. doi: 10.1002/j.1460-2075.1982.tb01167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Valentin-Hansen P., Holst B., Josephsen J., Hammer K., Albrechtsen B. CRP/cAMP- and CytR-regulated promoters in Escherichia coli K12: the cdd promoter. Mol Microbiol. 1989 Oct;3(10):1385–1390. doi: 10.1111/j.1365-2958.1989.tb00120.x. [DOI] [PubMed] [Google Scholar]
  30. Valentin-Hansen P., Larsen J. E., Højrup P., Short S. A., Barbier C. S. Nucleotide sequence of the CytR regulatory gene of E. coli K-12. Nucleic Acids Res. 1986 Mar 11;14(5):2215–2228. doi: 10.1093/nar/14.5.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Valentin-Hansen P. Tandem CRP binding sites in the deo operon of Escherichia coli K-12. EMBO J. 1982;1(9):1049–1054. doi: 10.1002/j.1460-2075.1982.tb01295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walton L., Richards C. A., Elwell L. P. Nucleotide sequence of the Escherichia coli uridine phosphorylase (udp) gene. Nucleic Acids Res. 1989 Aug 25;17(16):6741–6741. doi: 10.1093/nar/17.16.6741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  34. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]

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

RESOURCES