Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Nov;174(21):6807–6814. doi: 10.1128/jb.174.21.6807-6814.1992

ToxR proteins with substitutions in residues conserved with OmpR fail to activate transcription from the cholera toxin promoter.

K M Ottemann 1, V J DiRita 1, J J Mekalanos 1
PMCID: PMC207356  PMID: 1400230

Abstract

The ToxR protein of Vibrio cholerae is an integral membrane protein that coordinately regulates the expression of virulence genes required for successful infection. ToxR has been shown to bind directly to and activate transcription of the cholera toxin (ctx) promoter. Within the amino-terminal cytoplasmic region of ToxR, several amino acids are strictly conserved among ToxR, OmpR, and the other members of a family of bacterial regulatory proteins. To better understand the function of this region, two approaches were taken: conserved residues were changed by site-directed mutagenesis, and random mutations that eliminated ToxR-mediated transcriptional activation were isolated. Several classes of mutations were identified: those that abolish promoter DNA binding and transcriptional activation (toxR R96K, toxR R68K, and toxR R68L), those that abolish transcriptional activation but retain the ability to bind promoter DNA (toxR R96L), and those that have an intermediate phenotype (toxR R77L, toxR E51K, and toxR E51D). The toxR E51K allele had reduced activity in both Escherichia coli and V. cholerae but also exerted a dominant-negative effect over wild-type ToxR when assayed in V. cholerae. This result provides additional evidence that ToxR acts as an oligomer in the transcriptional activation process. From this mutational analysis of conserved amino acid residues within the OmpR-homologous region of ToxR, we conclude that this region is essential for transcriptional activation at the level of DNA binding and other steps that lead to activation of the ctx promoter.

Full text

PDF
6807

Images in this article

6811Image
on p.6811
6811Image
on p.6811
6812Image
on p.6812
6812Image
on p.6812

Selected References

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

  1. Albright L. M., Huala E., Ausubel F. M. Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu Rev Genet. 1989;23:311–336. doi: 10.1146/annurev.ge.23.120189.001523. [DOI] [PubMed] [Google Scholar]
  2. Bell A., Gaston K., Williams R., Chapman K., Kolb A., Buc H., Minchin S., Williams J., Busby S. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res. 1990 Dec 25;18(24):7243–7250. doi: 10.1093/nar/18.24.7243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Degnen G. E., Cox E. C. Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J Bacteriol. 1974 Feb;117(2):477–487. doi: 10.1128/jb.117.2.477-487.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DiRita V. J., Mekalanos J. J. Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation. Cell. 1991 Jan 11;64(1):29–37. doi: 10.1016/0092-8674(91)90206-e. [DOI] [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eschenlauer A. C., Reznikoff W. S. Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription. J Bacteriol. 1991 Aug;173(16):5024–5029. doi: 10.1128/jb.173.16.5024-5029.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Herrington D. A., Hall R. H., Losonsky G., Mekalanos J. J., Taylor R. K., Levine M. M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med. 1988 Oct 1;168(4):1487–1492. doi: 10.1084/jem.168.4.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holmgren J. Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect Immun. 1973 Dec;8(6):851–859. doi: 10.1128/iai.8.6.851-859.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hopkins J. D., Clements M. B., Liang T. Y., Isberg R. R., Syvanen M. Recombination genes on the Escherichia coli sex factor specific for transposable elements. Proc Natl Acad Sci U S A. 1980 May;77(5):2814–2818. doi: 10.1073/pnas.77.5.2814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Irwin N., Ptashne M. Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8315–8319. doi: 10.1073/pnas.84.23.8315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mekalanos J. J. Duplication and amplification of toxin genes in Vibrio cholerae. Cell. 1983 Nov;35(1):253–263. doi: 10.1016/0092-8674(83)90228-3. [DOI] [PubMed] [Google Scholar]
  18. Miller S. I., Kukral A. M., Mekalanos J. J. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5054–5058. doi: 10.1073/pnas.86.13.5054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller V. L., DiRita V. J., Mekalanos J. J. Identification of toxS, a regulatory gene whose product enhances toxR-mediated activation of the cholera toxin promoter. J Bacteriol. 1989 Mar;171(3):1288–1293. doi: 10.1128/jb.171.3.1288-1293.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller V. L., Mekalanos J. J. Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3471–3475. doi: 10.1073/pnas.81.11.3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller V. L., Taylor R. K., Mekalanos J. J. Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein. Cell. 1987 Jan 30;48(2):271–279. doi: 10.1016/0092-8674(87)90430-2. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Nara F., Inokuchi K., Matsuyama S., Mizushima S. Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli. J Bacteriol. 1984 Aug;159(2):688–692. doi: 10.1128/jb.159.2.688-692.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pearson G. D., Mekalanos J. J. Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1982 May;79(9):2976–2980. doi: 10.1073/pnas.79.9.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Peterson K. M., Mekalanos J. J. Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization. Infect Immun. 1988 Nov;56(11):2822–2829. doi: 10.1128/iai.56.11.2822-2829.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Raleigh E. A., Murray N. E., Revel H., Blumenthal R. M., Westaway D., Reith A. D., Rigby P. W., Elhai J., Hanahan D. McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res. 1988 Feb 25;16(4):1563–1575. doi: 10.1093/nar/16.4.1563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shinagawa H., Makino K., Nakata A. Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB. J Mol Biol. 1983 Aug 15;168(3):477–488. doi: 10.1016/s0022-2836(83)80297-6. [DOI] [PubMed] [Google Scholar]
  29. Slauch J. M., Russo F. D., Silhavy T. J. Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase. J Bacteriol. 1991 Dec;173(23):7501–7510. doi: 10.1128/jb.173.23.7501-7510.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  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. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taylor R. K., Miller V. L., Furlong D. B., Mekalanos J. J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci U S A. 1987 May;84(9):2833–2837. doi: 10.1073/pnas.84.9.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Watson N., Dunyak D. S., Rosey E. L., Slonczewski J. L., Olson E. R. Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH. J Bacteriol. 1992 Jan;174(2):530–540. doi: 10.1128/jb.174.2.530-540.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Williams R., Bell A., Sims G., Busby S. The role of two surface exposed loops in transcription activation by the Escherichia coli CRP and FNR proteins. Nucleic Acids Res. 1991 Dec 25;19(24):6705–6712. doi: 10.1093/nar/19.24.6705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamada M., Makino K., Amemura M., Shinagawa H., Nakata A. Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes. J Bacteriol. 1989 Oct;171(10):5601–5606. doi: 10.1128/jb.171.10.5601-5606.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES