Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Oct;176(20):6392–6396. doi: 10.1128/jb.176.20.6392-6396.1994

Maturation and localization of the TolB protein required for colicin import.

M Isnard 1, A Rigal 1, J C Lazzaroni 1, C Lazdunski 1, R Lloubes 1
PMCID: PMC196981  PMID: 7929011

Abstract

The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB*). To explain the presence of these two forms, two hypotheses have been proposed: TolB might be posttranslationally processed to TolB*, or an internal in-frame translation initiation resulting in TolB* may occur (S. K. Levengood and R. E. Webster, J. Bacteriol. 171:6600-6609, 1989). To address this question, TolB was tagged by inserting in its C-terminal region an epitope recognized by monoclonal antibody 1C11 without altering the function of TolB. It was then demonstrated that the functional protein corresponded to TolB*, the mature periplasmic protein, and that TolB was its precursor form, which was observed only when the protein was overexpressed. These two forms were purified by immunoprecipitation, and their N-terminal sequences were determined. An antibody directed against TolB was raised, which confirmed the results obtained with the tagged TolB.

Full text

PDF
6393

Images in this article

6393Image
on p.6393
6394Image
on p.6394
6394Image
on p.6394
6395Image
on p.6395
6395Image
on p.6395

Selected References

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

  1. Benedetti H., Lazdunski C., Lloubès R. Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system. EMBO J. 1991 Aug;10(8):1989–1995. doi: 10.1002/j.1460-2075.1991.tb07728.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolla J. M., Lazdunski C., Pagès J. M. The assembly of the major outer membrane protein OmpF of Escherichia coli depends on lipid synthesis. EMBO J. 1988 Nov;7(11):3595–3599. doi: 10.1002/j.1460-2075.1988.tb03237.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourdineaud J. P., Howard S. P., Lazdunski C. Localization and assembly into the Escherichia coli envelope of a protein required for entry of colicin A. J Bacteriol. 1989 May;171(5):2458–2465. doi: 10.1128/jb.171.5.2458-2465.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Cavard D., Crozel V., Gorvel J. P., Pattus F., Baty D., Lazdunski C. A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein. J Mol Biol. 1986 Feb 5;187(3):449–459. doi: 10.1016/0022-2836(86)90445-6. [DOI] [PubMed] [Google Scholar]
  6. Fognini-Lefebvre N., Lazzaroni J. C., Portalier R. tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12. Mol Gen Genet. 1987 Sep;209(2):391–395. doi: 10.1007/BF00329670. [DOI] [PubMed] [Google Scholar]
  7. Geli V., Lloubes R., Zaat S. A., van Spaendonk R. M., Rollin C., Benedetti H., Lazdunski C. Recognition of the colicin A N-terminal epitope 1C11 in vitro and in vivo in Escherichia coli by its cognate monoclonal antibody. FEMS Microbiol Lett. 1993 May 15;109(2-3):335–342. doi: 10.1016/0378-1097(93)90042-z. [DOI] [PubMed] [Google Scholar]
  8. Gorvel J. P., Ferrero A., Chambraud L., Rigal A., Bonicel J., Maroux S. Expression of sucrase-isomaltase and dipeptidylpeptidase IV in human small intestine and colon. Gastroenterology. 1991 Sep;101(3):618–625. doi: 10.1016/0016-5085(91)90517-o. [DOI] [PubMed] [Google Scholar]
  9. Guihard G., Boulanger P., Bénédetti H., Lloubés R., Besnard M., Letellier L. Colicin A and the Tol proteins involved in its translocation are preferentially located in the contact sites between the inner and outer membranes of Escherichia coli cells. J Biol Chem. 1994 Feb 25;269(8):5874–5880. [PubMed] [Google Scholar]
  10. Harboe N., Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl. 1973;1:161–164. doi: 10.1111/j.1365-3083.1973.tb03798.x. [DOI] [PubMed] [Google Scholar]
  11. Kampfenkel K., Braun V. Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the proposed first transmembrane segment. J Bacteriol. 1993 Jul;175(14):4485–4491. doi: 10.1128/jb.175.14.4485-4491.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Karlsson M., Hannavy K., Higgins C. F. A sequence-specific function for the N-terminal signal-like sequence of the TonB protein. Mol Microbiol. 1993 Apr;8(2):379–388. doi: 10.1111/j.1365-2958.1993.tb01581.x. [DOI] [PubMed] [Google Scholar]
  13. Kudlicki W., Kramer G., Hardesty B. High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system. Anal Biochem. 1992 Nov 1;206(2):389–393. doi: 10.1016/0003-2697(92)90383-i. [DOI] [PubMed] [Google Scholar]
  14. Lazzaroni J. C., Fognini-Lefebvre N., Portalier R. Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12. Mol Gen Genet. 1989 Sep;218(3):460–464. doi: 10.1007/BF00332410. [DOI] [PubMed] [Google Scholar]
  15. Levengood-Freyermuth S. K., Click E. M., Webster R. E. Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane. J Bacteriol. 1993 Jan;175(1):222–228. doi: 10.1128/jb.175.1.222-228.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levengood S. K., Beyer W. F., Jr, Webster R. E. TolA: a membrane protein involved in colicin uptake contains an extended helical region. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):5939–5943. doi: 10.1073/pnas.88.14.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Levengood S. K., Webster R. E. Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in Escherichia coli. J Bacteriol. 1989 Dec;171(12):6600–6609. doi: 10.1128/jb.171.12.6600-6609.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Muller M. M., Vianney A., Lazzaroni J. C., Webster R. E., Portalier R. Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity. J Bacteriol. 1993 Sep;175(18):6059–6061. doi: 10.1128/jb.175.18.6059-6061.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oliver D. B., Cabelli R. J., Dolan K. M., Jarosik G. P. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8227–8231. doi: 10.1073/pnas.87.21.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sun T. P., Webster R. E. Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli. J Bacteriol. 1987 Jun;169(6):2667–2674. doi: 10.1128/jb.169.6.2667-2674.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sun T. P., Webster R. E. fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB. J Bacteriol. 1986 Jan;165(1):107–115. doi: 10.1128/jb.165.1.107-115.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vianney A., Lewin T. M., Beyer W. F., Jr, Lazzaroni J. C., Portalier R., Webster R. E. Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity. J Bacteriol. 1994 Feb;176(3):822–829. doi: 10.1128/jb.176.3.822-829.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Webster R. E. The tol gene products and the import of macromolecules into Escherichia coli. Mol Microbiol. 1991 May;5(5):1005–1011. doi: 10.1111/j.1365-2958.1991.tb01873.x. [DOI] [PubMed] [Google Scholar]
  25. von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

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

RESOURCES