Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: Export of shiga-like toxin IIeB subunit by Salmonella typhimurium aroA

Nature Biotechnology volume 14, pages 765–769 (1996)Cite this article

Abstract

The export of Escherichia coli hemolysin across the cytoplasmic and the outer membranes requires the COOH-terminal signal sequence of HlyA, the two specific translocator proteins HlyB and HlyD, and the outer membrane protein TolC. We have developed an export cloning system that is composed of two vectors: one in which the fusion of the desired gene with the 3′-end of hlyA is generated, and a second in which the sequences containing the fusion are combined with the accessory genes hlyB and hlyD, thereby reconstructing the natural organization of the hly locus. In the second vector the fusion and the accessory genes are flanked by Not1 sites, allowing subcloning of the whole cluster into a variety of mini-transposons to achieve the stable integration of the constructs into the chromosome of Gram-negative bacteria. Since some applications may require the production of transcriptional fusions, an alternative version of the system provides the efficient translation initiation region of T7 phage gene 10 upstream of the fusion protein coding sequence. The usefulness of the system was assessed by constructing a fusion between the gene encoding the B subunit of Shiga-like toxin lie and the 3′-end of hlyA. An attenuated Salmonella typhimurium vaccine strain harboring the resulting construct, either in multicopy or mono-copy, efficiently expressed and exported the chimeric protein. We anticipate that this system will lead to a higher stability of the engineered function and permit a faithful monitoring of the export of the recombi-nant peptide under physiologic single-copy conditions.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Pugsley, A.P. 1993. The complete general secretion pathway in Gram-negative bacteria. Microbiol. Rev. 57: 50–108.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Holland, I.B., Wang, R., Seror, S.J., and Blight, M. Blight, M. 1990. Haemolysin secretion and other protein translocation mechanisms in gram-negative bacteria, pp. 219–254 in Microbial Products, New Approches. Baumberg, S., Hinster, I., and Rhodes, M. (eds.). Cambrige University Press, Cambridge, UK.

    Google Scholar 

  3. Wagner, W., Vogel, M., and Goebel, W. 1983. Transport of haemolysin across the outer membrane of Escherlchla coli requires two functions. J. Bacteriol. 154: 200–210.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Gray, L., Mackman, N., Nicaud, J.-M., and Holland, I.B. 1986. The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli . Mol. Gen. Genet. 205: 127–133.

    Article  CAS  PubMed  Google Scholar 

  5. Wandersman, C. and Delepelaire, P. 1990. TolC, an Escherichia coli outer membrane protein required for haemolysin secretion. Proc. Natl. Acad. Sci. USA 87: 4776–4780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Koronakis, V., Stanley, P., Koronakis, E., and Hughes, C. 1992. The HlyB/HlyD-dependent secretion of toxins by Gram-negative bacteria. FEMS Microbiol. Immunol. 105: 45–54.

    Article  Google Scholar 

  7. Holland, I.B., Kenny, B., and Blight, M. 1990. Haemolysin secretion from E. coli . Biochimie 72: 131–141.

    Article  CAS  PubMed  Google Scholar 

  8. Mackman, N., Baker, K., Gray, L., Haigh, R., Nicaud, J.M., and Holland, I.B. 1987. Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin. EMBO J. 6: 2835–2841.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hess, J., Gentschev, I., Goebel, W., and Jarchau, T. 1990. Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins. Mol. Gen. Genet. 224: 201–208.

    Article  CAS  PubMed  Google Scholar 

  10. Gentschev, I., Hess, J., and Goebel, W. 1990. Change in the cellular localization of alcaline phosphatase by alteration of its carboxy terminal sequence. Mol. Gen. Genet. 222: 211–216.

    Article  CAS  PubMed  Google Scholar 

  11. Gentschev, I., Sokolovic, Z., Köhler, S., Krohne, G.F., Hof, H., Wagner, J., and Goebel, W. 1992. Identification of p60 antibodies in human sera and presentation of this listerial antigen on the surface of attenuated Salmonellae by the HlyB-HlyD secretion system. Infect. Immun. 60: 5091–5098.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Su, G.-F., Brahmbhatt, H.N., Wehland, J., and Timmis, K.N. 1992. Construction of LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-speciflc mucosal and serum antibody responses. Infect. Immun. 60: 3345–3359.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Su, G.-F., Brahmbhatt, H.N., de Lorenzo, V., Wehland, J., and Timmis, K.N. 1992. Extracellular export of Shiga toxin B-subunit/haemolysin A (C-terminus) fusion protein expressed in Salmonella typhimurium aro/A-mutant and stimulation of B-subunit specific antibody responses in mice. Microb. Pathogen. 13: 465–476.

    Article  CAS  Google Scholar 

  14. Hashimoto-Gotoh, T., Franklin, F.C.H., Nordheim, A., and Timmis, K.N. 1981. Specific purpose plasmid cloning vectors: I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors. Gene 16: 227–235.

    Article  CAS  PubMed  Google Scholar 

  15. Karmali, M.A., Kausche, F.M., Dean, E.A., Arp, L.H., Samuel, J.E., and Moon, H.W. 1989. Infection by verotoxin-producing Escherichia coli . Clin. Microbiol. Rev. 2: 15–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wieler, L.H., Bauerfeind, R., and Baljer, G. 1992. Zur Bedeutung und Diagnostik von Infektionen landwirtschaftlicher Nutztiere mit Shiga Like Toxin produzieren-den E. coli (SLTEC). Tiërartzl. Umschau. 47: 524–533.

    Google Scholar 

  17. Weinstein, D.L., Jackson, M.P., Samuel, J.E., Holmes, R.K., and O'Brien, A.D. 1988. Cloning and sequencing of a single Shiga-Like Toxin typell variant from an Eschericha coli strain responsible for Edema disease of swine. J. Bacteriol. 170: 4223–4230.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kristensen, C.S., Eberl, L., Sanchez-Romero, J.M., Givskov, M., Molin, S., and de Lorenzo, V. 1994. Site specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host range plasmid RP4. J. Bacteriol. 177: 52–58.

    Article  Google Scholar 

  19. de Lorenzo, V. and Timmis, K.N. 1994. Analysis and construction of stable phe-notypes in Gram-negative bacteria with Tn5- and 7n10-derived minitransposons. Meth. Enzymol. 235: 386–405.

    Article  CAS  Google Scholar 

  20. Herrero, M., de Lorenzo, V., and Timmis, K.N. 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J. Bacteriol. 172: 6557–6567.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  22. Takeshita, S., Sato, M., Toba, M., Masahashi, W., and Hashimoto-Gotoh, T. 1987. High-copy number and low-copy number plasmid vectors for lacZα-complementation and chloramphenicol- or kanamycin-resistance selection. Gene 61: 63–74.

    Article  CAS  PubMed  Google Scholar 

  23. Felmlee, T., Pellett, S., and Welch, R.A. 1985. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J. Bacteriol. 163: 94–105.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Olinsa, P.O., Devine, C.S., Rangwala, S.H., and Kavka, K.S. 1988. The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli . Gene 73: 227–235.

    Article  Google Scholar 

  25. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 577–580.

    Article  Google Scholar 

  26. O'Callaghan, D. and Charbit, A. 1990. High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation. Mol. Gen. Genet. 223: 156–158.

    Article  CAS  PubMed  Google Scholar 

  27. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the heads of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  PubMed  Google Scholar 

  28. Towbin, H., Staehlin, H., and Gordon, G. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Richard, E. 1987. Preparation of genomic DNA from bacteria. Miniprep of bacterial chromosome DNA, pp. 1–2 in Current Protocols in Molecular Biology. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). John Wiley & Sons, Inc., New York.

    Google Scholar 

  30. Yanisch-Perron, C., Viera, J., and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119.

    Article  CAS  PubMed  Google Scholar 

  31. Mackman, N. and Holland, I.B. 1984. Functional characterisation of a cloned haemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107K polypeptide. Mol. Gen. Genet 193: 129–134.

    Article  Google Scholar 

  32. Nicaud, J.-M., Mackman, N., Gray, L., and Holland, I.B. 1986. The C-terminal, 23 kDa peptide of E. coli haemolysin 2001 contains all the information necessary for its secretion by the haemolysin (Hly) export machinery. FEBS Lett. 204: 331–335.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Carlos A. Guzmán: e-mail: CAG@gbf-braunschweig.de.

Authors and Affiliations

  1. Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, 38124, Braunschweig, Germany

    Barbara D. Tzschaschel, Carlos A. Guzmán, Kenneth N. Timmis & Victor de Lorenzo

  2. Centro de Investigaciones Biologicas CSIC, Velazquez 144, 28006, Madrid, Spain

    Victor de Lorenzo

Authors
  1. Barbara D. Tzschaschel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carlos A. Guzmán
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kenneth N. Timmis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Victor de Lorenzo
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tzschaschel, B., Guzmán, C., Timmis, K. et al. An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: Export of shiga-like toxin IIeB subunit by Salmonella typhimurium aroA. Nat Biotechnol 14, 765–769 (1996). https://doi.org/10.1038/nbt0696-765

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0696-765

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing