Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Aug;174(15):4907–4912. doi: 10.1128/jb.174.15.4907-4912.1992

Mutations in a central highly conserved non-DNA-binding region of OmpR, an Escherichia coli transcriptional activator, influence its DNA-binding ability.

R E Brissette 1, K Tsung 1, M Inouye 1
PMCID: PMC206302  PMID: 1321117

Abstract

OmpR is a transcriptional activator for the expression of outer membrane porin genes ompF and ompC in Escherichia coli. Its C-terminal half has been identified as the DNA-binding domain (K. Tsung, R. Brissette, and M. Inouye, J. Biol. Chem. 264:10104-10109, 1989). Recent studies have indicated that the N-terminal non-DNA-binding domain of OmpR is involved in modulating OmpR function through interaction with the EnvZ protein, a kinase and phosphatase for OmpR. We isolated and characterized two mutations, G94D and E111K, in the N-terminal domain of OmpR and one mutation, R182C, in the DNA-binding domain of OmpR. All three mutations abolished the ability of OmpR to bind to the ompF and ompC promoters in vivo, thus giving an OmpF- OmpC- phenotype. The decreased DNA-binding ability of the mutant OmpRs was not due to diminished phosphorylation of their N termini, since all the mutant OmpRs were found to be normally phosphorylated by EnvZ in vitro. The mutant OmpRs produced from multicopy plasmids were also found to inhibit completely the production of OmpF and OmpC in wild-type cells, and the complete inhibition depended on the function of EnvZ which was produced in cis or in trans from plasmids. The relationship of the possible alterations in OmpR by the mutations with the observed diminished binding ability is discussed.

Full text

PDF
4912

Images in this article

4908Image
on p.4908
4909Image
on p.4909
4909Image
on p.4909
4910Image
on p.4910
4910Image
on p.4910
4910Image
on p.4910

Selected References

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

  1. Aiba H., Mizuno T., Mizushima S. Transfer of phosphoryl group between two regulatory proteins involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem. 1989 May 25;264(15):8563–8567. [PubMed] [Google Scholar]
  2. Aiba H., Mizuno T. Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli. FEBS Lett. 1990 Feb 12;261(1):19–22. doi: 10.1016/0014-5793(90)80626-t. [DOI] [PubMed] [Google Scholar]
  3. Aiba H., Nakasai F., Mizushima S., Mizuno T. Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability. J Biochem. 1989 Jul;106(1):5–7. doi: 10.1093/oxfordjournals.jbchem.a122817. [DOI] [PubMed] [Google Scholar]
  4. Alphen W. V., Lugtenberg B. Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli. J Bacteriol. 1977 Aug;131(2):623–630. doi: 10.1128/jb.131.2.623-630.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brissette R. E., Tsung K. L., Inouye M. Intramolecular second-site revertants to the phosphorylation site mutation in OmpR, a kinase-dependent transcriptional activator in Escherichia coli. J Bacteriol. 1991 Jun;173(12):3749–3755. doi: 10.1128/jb.173.12.3749-3755.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brissette R. E., Tsung K. L., Inouye M. Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli. J Bacteriol. 1991 Jan;173(2):601–608. doi: 10.1128/jb.173.2.601-608.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Forst S., Delgado J., Inouye M. Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6052–6056. doi: 10.1073/pnas.86.16.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Ikenaka K., Tsung K., Comeau D. E., Inouye M. A dominant mutation in Escherichia coli OmpR lies within a domain which is highly conserved in a large family of bacterial regulatory proteins. Mol Gen Genet. 1988 Mar;211(3):538–540. doi: 10.1007/BF00425713. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Mizuno T., Kato M., Jo Y. L., Mizushima S. Interaction of OmpR, a positive regulator, with the osmoregulated ompC and ompF genes of Escherichia coli. Studies with wild-type and mutant OmpR proteins. J Biol Chem. 1988 Jan 15;263(2):1008–1012. [PubMed] [Google Scholar]
  19. Nakashima K., Kanamaru K., Aiba H., Mizuno T. Signal transduction and osmoregulation in Escherichia coli. A novel type of mutation in the phosphorylation domain of the activator protein, OmpR, results in a defect in its phosphorylation-dependent DNA binding. J Biol Chem. 1991 Jun 15;266(17):10775–10780. [PubMed] [Google Scholar]
  20. Nara F., Mizuno T., Mizushima S. Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli. Mol Gen Genet. 1986 Oct;205(1):51–55. [PubMed] [Google Scholar]
  21. 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]
  22. Rose R. E. The nucleotide sequence of pACYC184. Nucleic Acids Res. 1988 Jan 11;16(1):355–355. doi: 10.1093/nar/16.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanders D. A., Gillece-Castro B. L., Stock A. M., Burlingame A. L., Koshland D. E., Jr Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. J Biol Chem. 1989 Dec 25;264(36):21770–21778. [PubMed] [Google Scholar]
  24. 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]
  25. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stock J. B., Stock A. M., Mottonen J. M. Signal transduction in bacteria. Nature. 1990 Mar 29;344(6265):395–400. doi: 10.1038/344395a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Tsung K., Brissette R. E., Inouye M. Identification of the DNA-binding domain of the OmpR protein required for transcriptional activation of the ompF and ompC genes of Escherichia coli by in vivo DNA footprinting. J Biol Chem. 1989 Jun 15;264(17):10104–10109. [PubMed] [Google Scholar]
  29. Utsumi R., Brissette R. E., Rampersaud A., Forst S. A., Oosawa K., Inouye M. Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate. Science. 1989 Sep 15;245(4923):1246–1249. doi: 10.1126/science.2476847. [DOI] [PubMed] [Google Scholar]

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

RESOURCES