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. 1997 Jun;179(11):3683–3690. doi: 10.1128/jb.179.11.3683-3690.1997

The TolA protein interacts with colicin E1 differently than with other group A colicins.

S L Schendel 1, E M Click 1, R E Webster 1, W A Cramer 1
PMCID: PMC179165  PMID: 9171417

Abstract

The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability. Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.

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Selected References

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  1. Benedetti H., Frenette M., Baty D., Knibiehler M., Pattus F., Lazdunski C. Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement. J Mol Biol. 1991 Feb 5;217(3):429–439. doi: 10.1016/0022-2836(91)90747-t. [DOI] [PubMed] [Google Scholar]
  2. Benedetti H., Frenette M., Baty D., Lloubès R., Geli V., Lazdunski C. Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coli. J Gen Microbiol. 1989 Dec;135(12):3413–3420. doi: 10.1099/00221287-135-12-3413. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bernstein A., Rolfe B., Onodera K. Pleiotropic properties and genetic organization of the tolA,B locus of Escherichia coli K-12. J Bacteriol. 1972 Oct;112(1):74–83. doi: 10.1128/jb.112.1.74-83.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braun V., Pilsl H., Gross P. Colicins: structures, modes of action, transfer through membranes, and evolution. Arch Microbiol. 1994;161(3):199–206. doi: 10.1007/BF00248693. [DOI] [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. Brunden K. R., Cramer W. A., Cohen F. S. Purification of a small receptor-binding peptide from the central region of the colicin E1 molecule. J Biol Chem. 1984 Jan 10;259(1):190–196. [PubMed] [Google Scholar]
  8. Bullock J. O., Cohen F. S., Dankert J. R., Cramer W. A. Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide. J Biol Chem. 1983 Aug 25;258(16):9908–9912. [PubMed] [Google Scholar]
  9. Bénédetti H., Lloubès R., Lazdunski C., Letellier L. Colicin A unfolds during its translocation in Escherichia coli cells and spans the whole cell envelope when its pore has formed. EMBO J. 1992 Feb;11(2):441–447. doi: 10.1002/j.1460-2075.1992.tb05073.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cavard D., Lazdunski C. J. Purification and molecular properties of a new colicin. Eur J Biochem. 1979 Jun 1;96(3):519–524. doi: 10.1111/j.1432-1033.1979.tb13065.x. [DOI] [PubMed] [Google Scholar]
  11. Cramer W. A., Heymann J. B., Schendel S. L., Deriy B. N., Cohen F. S., Elkins P. A., Stauffacher C. V. Structure-function of the channel-forming colicins. Annu Rev Biophys Biomol Struct. 1995;24:611–641. doi: 10.1146/annurev.bb.24.060195.003143. [DOI] [PubMed] [Google Scholar]
  12. Dankert J. R., Uratani Y., Grabau C., Cramer W. A., Hermodson M. On a domain structure of colicin E1. A COOH-terminal peptide fragment active in membrane depolarization. J Biol Chem. 1982 Apr 10;257(7):3857–3863. [PubMed] [Google Scholar]
  13. Derouiche R., Bénédetti H., Lazzaroni J. C., Lazdunski C., Lloubès R. Protein complex within Escherichia coli inner membrane. TolA N-terminal domain interacts with TolQ and TolR proteins. J Biol Chem. 1995 May 12;270(19):11078–11084. doi: 10.1074/jbc.270.19.11078. [DOI] [PubMed] [Google Scholar]
  14. Derouiche R., Gavioli M., Bénédetti H., Prilipov A., Lazdunski C., Lloubès R. TolA central domain interacts with Escherichia coli porins. EMBO J. 1996 Dec 2;15(23):6408–6415. [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Jakes K. S., Zinder N. D. Highly purified colicin E3 contains immunity protein. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3380–3384. doi: 10.1073/pnas.71.9.3380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jaskula J. C., Letain T. E., Roof S. K., Skare J. T., Postle K. Role of the TonB amino terminus in energy transduction between membranes. J Bacteriol. 1994 Apr;176(8):2326–2338. doi: 10.1128/jb.176.8.2326-2338.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Kellenberger E. The 'Bayer bridges' confronted with results from improved electron microscopy methods. Mol Microbiol. 1990 May;4(5):697–705. doi: 10.1111/j.1365-2958.1990.tb00640.x. [DOI] [PubMed] [Google Scholar]
  20. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Larsen R. A., Wood G. E., Postle K. The conserved proline-rich motif is not essential for energy transduction by Escherichia coli TonB protein. Mol Microbiol. 1994 Jun;12(5):857–857. doi: 10.1111/j.1365-2958.1994.tb01072.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lazzaroni J. C., Vianney A., Popot J. L., Bénédetti H., Samatey F., Lazdunski C., Portalier R., Géli V. Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex. J Mol Biol. 1995 Feb 10;246(1):1–7. doi: 10.1006/jmbi.1994.0058. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Marqusee S., Baldwin R. L. Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8898–8902. doi: 10.1073/pnas.84.24.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mel S. F., Stroud R. M. Colicin Ia inserts into negatively charged membranes at low pH with a tertiary but little secondary structural change. Biochemistry. 1993 Mar 2;32(8):2082–2089. doi: 10.1021/bi00059a028. [DOI] [PubMed] [Google Scholar]
  29. Morona R., Manning P. A., Reeves P. Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli. J Bacteriol. 1983 Feb;153(2):693–699. doi: 10.1128/jb.153.2.693-699.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Nagel de Zwaig R., Luria S. E. Genetics and physiology of colicin-tolerant mutants of Escherichia coli. J Bacteriol. 1967 Oct;94(4):1112–1123. doi: 10.1128/jb.94.4.1112-1123.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nikaido H. Porins and specific channels of bacterial outer membranes. Mol Microbiol. 1992 Feb;6(4):435–442. doi: 10.1111/j.1365-2958.1992.tb01487.x. [DOI] [PubMed] [Google Scholar]
  33. Schatz G., Dobberstein B. Common principles of protein translocation across membranes. Science. 1996 Mar 15;271(5255):1519–1526. doi: 10.1126/science.271.5255.1519. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Song H. Y., Cohen F. S., Cramer W. A. Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin. J Bacteriol. 1991 May;173(9):2927–2934. doi: 10.1128/jb.173.9.2927-2934.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Van Wielink J. E., Duine J. A. How big is the periplasmic space? Trends Biochem Sci. 1990 Apr;15(4):136–137. doi: 10.1016/0968-0004(90)90208-s. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Vianney A., Muller M. M., Clavel T., Lazzaroni J. C., Portalier R., Webster R. E. Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein. J Bacteriol. 1996 Jul;178(14):4031–4038. doi: 10.1128/jb.178.14.4031-4038.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wandersman C., Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4776–4780. doi: 10.1073/pnas.87.12.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Zhang L. H., Fath M. J., Mahanty H. K., Tai P. C., Kolter R. Genetic analysis of the colicin V secretion pathway. Genetics. 1995 Sep;141(1):25–32. doi: 10.1093/genetics/141.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang Y. L., Cramer W. A. Intramembrane helix-helix interactions as the basis of inhibition of the colicin E1 ion channel by its immunity protein. J Biol Chem. 1993 May 15;268(14):10176–10184. [PubMed] [Google Scholar]

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