Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Feb;176(3):822–829. doi: 10.1128/jb.176.3.822-829.1994

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

A Vianney 1, T M Lewin 1, W F Beyer Jr 1, J C Lazzaroni 1, R Portalier 1, R E Webster 1
PMCID: PMC205120  PMID: 8300535

Abstract

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.

Full text

PDF
822

Images in this article

825Image
on p.825
826Image
on p.826
826Image
on p.826
827Image
on p.827

Selected References

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

  1. Barany F. Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering. Gene. 1985;37(1-3):111–123. doi: 10.1016/0378-1119(85)90263-x. [DOI] [PubMed] [Google Scholar]
  2. Barany F. Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4202–4206. doi: 10.1073/pnas.82.12.4202. [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. Braun V., Herrmann C. Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: cross-complementation between the TonB-ExbB-ExbD and the TolA-TolQ-TolR proteins. Mol Microbiol. 1993 Apr;8(2):261–268. doi: 10.1111/j.1365-2958.1993.tb01570.x. [DOI] [PubMed] [Google Scholar]
  5. Braun V. The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity. J Bacteriol. 1989 Nov;171(11):6387–6390. doi: 10.1128/jb.171.11.6387-6390.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brewer S., Tolley M., Trayer I. P., Barr G. C., Dorman C. J., Hannavy K., Higgins C. F., Evans J. S., Levine B. A., Wormald M. R. Structure and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella typhimurium and Escherichia coli. J Mol Biol. 1990 Dec 20;216(4):883–895. doi: 10.1016/S0022-2836(99)80008-4. [DOI] [PubMed] [Google Scholar]
  7. Eick-Helmerich K., Braun V. Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively. J Bacteriol. 1989 Sep;171(9):5117–5126. doi: 10.1128/jb.171.9.5117-5126.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gutierrez C., Devedjian J. C. A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3999–3999. doi: 10.1093/nar/17.10.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guy-Caffey J. K., Rapoza M. P., Jolley K. A., Webster R. E. Membrane localization and topology of a viral assembly protein. J Bacteriol. 1992 Apr;174(8):2460–2465. doi: 10.1128/jb.174.8.2460-2465.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Kampfenkel K., Braun V. Membrane topology of the Escherichia coli ExbD protein. J Bacteriol. 1992 Aug;174(16):5485–5487. doi: 10.1128/jb.174.16.5485-5487.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kampfenkel K., Braun V. Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli. J Biol Chem. 1993 Mar 15;268(8):6050–6057. [PubMed] [Google Scholar]
  13. 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]
  14. Karlsson M., Hannavy K., Higgins C. F. ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm. Mol Microbiol. 1993 Apr;8(2):389–396. doi: 10.1111/j.1365-2958.1993.tb01582.x. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lazzaroni J. C., Portalier R. C. Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12. J Bacteriol. 1981 Mar;145(3):1351–1358. doi: 10.1128/jb.145.3.1351-1358.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lazzaroni J. C., Portalier R. The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein (PAL). Mol Microbiol. 1992 Mar;6(6):735–742. doi: 10.1111/j.1365-2958.1992.tb01523.x. [DOI] [PubMed] [Google Scholar]
  19. Lee J. I., Kuhn A., Dalbey R. E. Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion. J Biol Chem. 1992 Jan 15;267(2):938–943. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Manoil C. Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase gene fusions. Methods Cell Biol. 1991;34:61–75. doi: 10.1016/s0091-679x(08)61676-3. [DOI] [PubMed] [Google Scholar]
  24. Manoil C. Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol. 1990 Feb;172(2):1035–1042. doi: 10.1128/jb.172.2.1035-1042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Palmiter R. D., Gagnon J., Walsh K. A. Ovalbumin: a secreted protein without a transient hydrophobic leader sequence. Proc Natl Acad Sci U S A. 1978 Jan;75(1):94–98. doi: 10.1073/pnas.75.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Postle K. TonB and the gram-negative dilemma. Mol Microbiol. 1990 Dec;4(12):2019–2025. doi: 10.1111/j.1365-2958.1990.tb00561.x. [DOI] [PubMed] [Google Scholar]
  29. Roof S. K., Allard J. D., Bertrand K. P., Postle K. Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions. J Bacteriol. 1991 Sep;173(17):5554–5557. doi: 10.1128/jb.173.17.5554-5557.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Skare J. T., Postle K. Evidence for a TonB-dependent energy transduction complex in Escherichia coli. Mol Microbiol. 1991 Dec;5(12):2883–2890. doi: 10.1111/j.1365-2958.1991.tb01848.x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Valentin-Hansen P., Albrechtsen B., Løve Larsen J. E. DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. EMBO J. 1986 Aug;5(8):2015–2021. doi: 10.1002/j.1460-2075.1986.tb04458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Webster R. E., Engelhardt D. L., Zinder N. D. In vitro protein synthesis: chain initiation. Proc Natl Acad Sci U S A. 1966 Jan;55(1):155–161. doi: 10.1073/pnas.55.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Wolfe P. B., Wickner W., Goodman J. M. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope. J Biol Chem. 1983 Oct 10;258(19):12073–12080. [PubMed] [Google Scholar]
  36. von Heijne G., Wickner W., Dalbey R. E. The cytoplasmic domain of Escherichia coli leader peptidase is a "translocation poison" sequence. Proc Natl Acad Sci U S A. 1988 May;85(10):3363–3366. doi: 10.1073/pnas.85.10.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES