Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1986 Oct;168(1):86–95. doi: 10.1128/jb.168.1.86-95.1986

Characterization by deletion and localized mutagenesis in vitro of the promoter region of the Escherichia coli ompC gene and importance of the upstream DNA domain in positive regulation by the OmpR protein.

T Mizuno, S Mizushima
PMCID: PMC213423  PMID: 3531187

Abstract

The ompC gene codes for a major outer membrane protein whose expression is regulated by the ompR and envZ genes. Two sets of promoter deletion mutants, with upstream and downstream deletions, were constructed on a plasmid in vitro, and their promoter activity was studied by connecting them with the lacZ gene. The DNA sequence for the ompC promoter, including the -35 and -10 regions and the mRNA start site, was defined at the region about 100 base pairs upstream from the ATG initiation codon for the pro-OmpC protein. An additional 61-base-pair sequence extending upstream from the -35 region was required for the ompC promoter to function fully. After targeting the upstream region of the ompC promoter fused to the lacZ gene on a plasmid, in vitro-localized mutagenesis was performed to isolate cis-dominant mutations that affect ompC transcription. Four mutant groups, each of which had common phenotypes for expression and regulation of the gene, were identified. The individual groups also had common base substitutions. In two of the groups, the common base substitutions were localized in the upstream region of the ompC promoter, whereas in the other two they were localized in the -35 region. From these results, the upstream region of the ompC promoter was considered to be the domain responsible for activation by the ompR gene product.

Full text

PDF
89

Selected References

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

  1. 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]
  2. Baas P. D., van Teeffelen H. A., Teertstra W. R., Jansz H. S., Veeneman G. H., van der Marel G. A., van Boom J. H. Restoration of the biological activity of in vitro synthesized phi X DNA by transfection of ung- spheroplasts or dUTPase treatment. FEBS Lett. 1980 Jan 28;110(1):15–20. doi: 10.1016/0014-5793(80)80012-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Dairi T., Inokuchi K., Mizuno T., Mizushima S. Positive control of transcription initiation in Escherichia coli. A base substitution at the Pribnow box renders ompF expression independent of a positive regulator. J Mol Biol. 1985 Jul 5;184(1):1–6. doi: 10.1016/0022-2836(85)90038-5. [DOI] [PubMed] [Google Scholar]
  5. Hall M. N., Silhavy T. J. Genetic analysis of the major outer membrane proteins of Escherichia coli. Annu Rev Genet. 1981;15:91–142. doi: 10.1146/annurev.ge.15.120181.000515. [DOI] [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. 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]
  10. Inokuchi K., Furukawa H., Nakamura K., Mizushima S. Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli. J Mol Biol. 1984 Sep 25;178(3):653–668. doi: 10.1016/0022-2836(84)90243-2. [DOI] [PubMed] [Google Scholar]
  11. Inokuchi K., Itoh M., Mizushima S. Domains involved in osmoregulation of the ompF gene in Escherichia coli. J Bacteriol. 1985 Nov;164(2):585–590. doi: 10.1128/jb.164.2.585-590.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inokuchi K., Mutoh N., Matsuyama S., Mizushima S. Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12. Nucleic Acids Res. 1982 Nov 11;10(21):6957–6968. doi: 10.1093/nar/10.21.6957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kalderon D., Oostra B. A., Ely B. K., Smith A. E. Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants. Nucleic Acids Res. 1982 Sep 11;10(17):5161–5171. doi: 10.1093/nar/10.17.5161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mizuno T., Chou M. Y., Inouye M. A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene. J Biol Chem. 1983 Jun 10;258(11):6932–6940. [PubMed] [Google Scholar]
  17. Mizuno T., Chou M. Y., Inouye M. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A. 1984 Apr;81(7):1966–1970. doi: 10.1073/pnas.81.7.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mizuno T., Chou M. Y., Inouye M. DNA sequence of the promoter region of the ompC gene and the amino acid sequence of the signal peptide of pro-OmpC protein of Escherichia coli. FEBS Lett. 1983 Jan 10;151(1):159–164. doi: 10.1016/0014-5793(83)80364-0. [DOI] [PubMed] [Google Scholar]
  19. Mizuno T., Wurtzel E. T., Inouye M. Cloning of the regulatory genes (ompR and envZ) for the matrix proteins of the Escherichia coli outer membrane. J Bacteriol. 1982 Jun;150(3):1462–1466. doi: 10.1128/jb.150.3.1462-1466.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Nara F., Matsuyama S., Mizuno T., Mizushima S. Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli. Mol Gen Genet. 1986 Feb;202(2):194–199. doi: 10.1007/BF00331636. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Shortle D., Koshland D., Weinstock G. M., Botstein D. Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5375–5379. doi: 10.1073/pnas.77.9.5375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Taylor R. K., Hall M. N., Enquist L., Silhavy T. J. Identification of OmpR: a positive regulatory protein controlling expression of the major outer membrane matrix porin proteins of Escherichia coli K-12. J Bacteriol. 1981 Jul;147(1):255–258. doi: 10.1128/jb.147.1.255-258.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Taylor R. K., Hall M. N., Silhavy T. J. Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine. J Mol Biol. 1983 May 25;166(3):273–282. doi: 10.1016/s0022-2836(83)80085-0. [DOI] [PubMed] [Google Scholar]
  27. Wurtzel E. T., Chou M. Y., Inouye M. Osmoregulation of gene expression. I. DNA sequence of the ompR gene of the ompB operon of Escherichia coli and characterization of its gene product. J Biol Chem. 1982 Nov 25;257(22):13685–13691. [PubMed] [Google Scholar]

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

RESOURCES