Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Aug;86(16):6052–6056. doi: 10.1073/pnas.86.16.6052

Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli.

S Forst 1, J Delgado 1, M Inouye 1
PMCID: PMC297773  PMID: 2668953

Abstract

EnvZ and OmpR, the regulatory proteins for ompF and ompC expression in Escherichia coli, belong to a modulator-effector family of regulatory proteins which are essential for the response to environmental signals. We show that the soluble cytoplasmic domain of the transmembrane modulator protein EnvZ is phosphorylated in vitro by [gamma-32P]-ATP. We also demonstrate that the phosphate group can, in turn, be transferred to the transcription activator protein OmpR. The pH stability properties of the phosphate groups linked to EnvZ indicate that this molecule contains histidyl phosphate. The invariant His-243 of EnvZ corresponds to the phosphorylated His-48 of the chemotactic modulator protein CheA. Substitution of His-243 with valine produces an EnvZ that is refractory to phosphorylation and can no longer catalyze the transfer of phosphate to OmpR. Furthermore, in a delta envZ strain of E. coli, containing the envZ Val-243 plasmid, ompC expression is elevated 7-fold relative to that found in cells carrying the wild-type envZ plasmid. Based on these results we propose a model in which the phosphorylated state of OmpR modulates the expression of the ompF and ompC genes.

Full text

PDF
6053

Images in this article

6053Image
on p.6053
6053Image
on p.6053
6053Image
on p.6053
6054Image
on p.6054
6054Image
on p.6054
6054Image
on p.6054
6055Image
on p.6055

Selected References

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

  1. Comeau D. E., Ikenaka K., Tsung K. L., Inouye M. Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins. J Bacteriol. 1985 Nov;164(2):578–584. doi: 10.1128/jb.164.2.578-584.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Forst S., Comeau D., Norioka S., Inouye M. Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli. J Biol Chem. 1987 Dec 5;262(34):16433–16438. [PubMed] [Google Scholar]
  3. Forst S., Delgado J., Ramakrishnan G., Inouye M. Regulation of ompC and ompF expression in Escherichia coli in the absence of envZ. J Bacteriol. 1988 Nov;170(11):5080–5085. doi: 10.1128/jb.170.11.5080-5085.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Forst S., Inouye M. Environmentally regulated gene expression for membrane proteins in Escherichia coli. Annu Rev Cell Biol. 1988;4:21–42. doi: 10.1146/annurev.cb.04.110188.000321. [DOI] [PubMed] [Google Scholar]
  5. Garrett S., Taylor R. K., Silhavy T. J., Berman M. L. Isolation and characterization of delta ompB strains of Escherichia coli by a general method based on gene fusions. J Bacteriol. 1985 May;162(2):840–844. doi: 10.1128/jb.162.2.840-844.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hall M. N., Silhavy T. J. Genetic analysis of the ompB locus in Escherichia coli K-12. J Mol Biol. 1981 Sep 5;151(1):1–15. doi: 10.1016/0022-2836(81)90218-7. [DOI] [PubMed] [Google Scholar]
  7. Hall M. N., Silhavy T. J. The ompB locus and the regulation of the major outer membrane porin proteins of Escherichia coli K12. J Mol Biol. 1981 Feb 15;146(1):23–43. doi: 10.1016/0022-2836(81)90364-8. [DOI] [PubMed] [Google Scholar]
  8. Hall M. N., Silhavy T. J. Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b. J Bacteriol. 1979 Nov;140(2):342–350. doi: 10.1128/jb.140.2.342-350.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hess J. F., Bourret R. B., Simon M. I. Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. Nature. 1988 Nov 10;336(6195):139–143. doi: 10.1038/336139a0. [DOI] [PubMed] [Google Scholar]
  10. Hess J. F., Oosawa K., Kaplan N., Simon M. I. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell. 1988 Apr 8;53(1):79–87. doi: 10.1016/0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  11. Igo M. M., Ninfa A. J., Silhavy T. J. A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation. Genes Dev. 1989 May;3(5):598–605. doi: 10.1101/gad.3.5.598. [DOI] [PubMed] [Google Scholar]
  12. Igo M. M., Silhavy T. J. EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro. J Bacteriol. 1988 Dec;170(12):5971–5973. doi: 10.1128/jb.170.12.5971-5973.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ikenaka K., Ramakrishnan G., Inouye M., Tsung K., Inouye M. Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters. J Biol Chem. 1986 Jul 15;261(20):9316–9320. [PubMed] [Google Scholar]
  14. Inouye S., Inouye M. Up-promoter mutations in the lpp gene of Escherichia coli. Nucleic Acids Res. 1985 May 10;13(9):3101–3110. doi: 10.1093/nar/13.9.3101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jo Y. L., Nara F., Ichihara S., Mizuno T., Mizushima S. Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem. 1986 Nov 15;261(32):15252–15256. [PubMed] [Google Scholar]
  16. Kawaji H., Mizuno T., Mizushima S. Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K-12. J Bacteriol. 1979 Dec;140(3):843–847. doi: 10.1128/jb.140.3.843-847.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keener J., Kustu S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976–4980. doi: 10.1073/pnas.85.14.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Martensen T. M. Chemical properties, isolation, and analysis of O-phosphates in proteins. Methods Enzymol. 1984;107:3–23. doi: 10.1016/0076-6879(84)07003-8. [DOI] [PubMed] [Google Scholar]
  19. Matsuyama S., Mizuno T., Mizushima S. Interaction between two regulatory proteins in osmoregulatory expression of ompF and ompC genes in Escherichia coli: a novel ompR mutation suppresses pleiotropic defects caused by an envZ mutation. J Bacteriol. 1986 Dec;168(3):1309–1314. doi: 10.1128/jb.168.3.1309-1314.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mizuno T., Mizushima S. Isolation and characterization of deletion mutants of ompR and envZ, regulatory genes for expression of the outer membrane proteins OmpC and OmpF in Escherichia coli. J Biochem. 1987 Feb;101(2):387–396. doi: 10.1093/oxfordjournals.jbchem.a121923. [DOI] [PubMed] [Google Scholar]
  21. Mizuno T. Static bend of DNA helix at the activator recognition site of the ompF promoter in Escherichia coli. Gene. 1987;54(1):57–64. doi: 10.1016/0378-1119(87)90347-7. [DOI] [PubMed] [Google Scholar]
  22. Mizuno T., Wurtzel E. T., Inouye M. Osmoregulation of gene expression. II. DNA sequence of the envZ gene of the ompB operon of Escherichia coli and characterization of its gene product. J Biol Chem. 1982 Nov 25;257(22):13692–13698. [PubMed] [Google Scholar]
  23. Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ninfa A. J., Ninfa E. G., Lupas A. N., Stock A., Magasanik B., Stock J. Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5492–5496. doi: 10.1073/pnas.85.15.5492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nixon B. T., Ronson C. W., Ausubel F. M. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7850–7854. doi: 10.1073/pnas.83.20.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Norioka S., Ramakrishnan G., Ikenaka K., Inouye M. Interaction of a transcriptional activator, OmpR, with reciprocally osmoregulated genes, ompF and ompC, of Escherichia coli. J Biol Chem. 1986 Dec 25;261(36):17113–17119. [PubMed] [Google Scholar]
  27. Sarma V., Reeves P. Genetic locus (ompB) affecting a major outer-membrane protein in Escherichia coli K-12. J Bacteriol. 1977 Oct;132(1):23–27. doi: 10.1128/jb.132.1.23-27.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stock A. M., Mottonen J. M., Stock J. B., Schutt C. E. Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis. Nature. 1989 Feb 23;337(6209):745–749. doi: 10.1038/337745a0. [DOI] [PubMed] [Google Scholar]
  29. Stock A. M., Wylie D. C., Mottonen J. M., Lupas A. N., Ninfa E. G., Ninfa A. J., Schutt C. E., Stock J. B. Phosphoproteins involved in bacterial signal transduction. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 1):49–57. doi: 10.1101/sqb.1988.053.01.009. [DOI] [PubMed] [Google Scholar]
  30. Stock A., Chen T., Welsh D., Stock J. CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1403–1407. doi: 10.1073/pnas.85.5.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tate S., Kato M., Nishimura Y., Arata Y., Mizuno T. Location of DNA-binding segment of a positive regulator, OmpR, involved in activation of the ompF and ompC genes of Escherichia coli. FEBS Lett. 1988 Dec 19;242(1):27–30. doi: 10.1016/0014-5793(88)80978-5. [DOI] [PubMed] [Google Scholar]
  32. Verhoef C., Lugtenberg B., van Boxtel R., de Graaff P., Verheij H. Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12. Mol Gen Genet. 1979 Jan 31;169(2):137–146. doi: 10.1007/BF00271664. [DOI] [PubMed] [Google Scholar]
  33. Weiss V., Magasanik B. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919–8923. doi: 10.1073/pnas.85.23.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wylie D., Stock A., Wong C. Y., Stock J. Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins. Biochem Biophys Res Commun. 1988 Mar 15;151(2):891–896. doi: 10.1016/s0006-291x(88)80365-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES