Molecular and General Genetics MGG

, Volume 245, Issue 1, pp 53–60 | Cite as

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

  • T. Jarchau
  • T. Chakraborty
  • F. Garcia
  • W. Goebel
Original Paper
Received:
Accepted:

Abstract

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5′ end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

Key words

Escherichia coli hemolysin Transport Signal sequence Peptide transport 
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References

  1. Cleveland DW (1983) Peptide mapping in one dimension by limited proteolysis of sodium dodecyl sulfate-solubilized proteins. Methods Enzymol 96:222–229Google Scholar
  2. Coote JG (1992) Structural and functional relationships among the RTX toxin determinants of gram-negative bacteria. FEMS Microbiol Rev 88:137–162Google Scholar
  3. Delepelaire P, Wandersman C (1990) Protein secretion in gram-negative bacteria: the extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli α-hemolysin. J Biol Chem 265:17:118–125Google Scholar
  4. Economou AE, Hamilton WDO, Johnston AWB, Downie JA (1990) The rhizobium modulation gene nodO encodes a Ca2+binding protein that is exported without an N-terminal cleavage and is homologous to hemolysin and related proteins. EMBO J 9:349–354Google Scholar
  5. Fath MJ, Skvirsky RC, Kolter R (1991) Functional complementation between bacterial MDR-like export systems: colicin V, α-hemolysin, and Erwinia protease. J Bacteriol 173:7549–7556Google Scholar
  6. Felmlee T, Welch RA (1988) Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc Natl Acad Sci USA 85:5269–5273Google Scholar
  7. Felmlee T, Pellett S, Lee E-Y, Welch RA (1985a) Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J Bacteriol 163:88–93Google Scholar
  8. Felmlee T, Pellett S, Welch RA (1985b) Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol 163:94–105Google Scholar
  9. Ferenci T, Silhavy TJ (1987) Sequence information required for protein translocation from the cytoplasm. J Bacteriol 169:5339–5342Google Scholar
  10. Gentschev I, Hess J, Goebel W (1990) Change in the cellular localization of alkaline phosphatase by alteration of its carboxyterminal sequence. Mol Gen Genet 222:211–216Google Scholar
  11. Guzzo J, Duong F, Wandersman C, Murgier M, Lazdunski A (1991) The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli α-hemolysin. Mol Microbiol 5:447–453Google Scholar
  12. Hanke C, Hess J, Schumacher G, Goebel W (1992) Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system. Mol Gen Genet 233:42–48Google Scholar
  13. Härtlein M, Schiessl S, Wagner W, Rdest U, Kreft J, Goebel W (1983) Transport of hemolysin by Escherichia coli. J Cell Biochem 22:87–97Google Scholar
  14. Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142–147Google Scholar
  15. Henikoff S (1987) Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol 155:156–165Google Scholar
  16. Hess J, Wels W, Vogel M, Goebel W (1986) Nucleotide sequence of a plasmid-encoded hemolysin determinant and its comparison with a corresponding chromosonal hemolysin sequence. FEMS Microbiol Lett 34:1–11Google Scholar
  17. Hess J, Gentschev I, Goebel W, Jarchau T (1990) Analysis of the hemolysin secretion system by PhoA-HlyA fusion proteins. Mol Gen Genet 244:201–208Google Scholar
  18. Kenny B, Haig R, Holland IB (1991) Analysis of the hemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β-galactosidase fused to the HlyA C-terminal domain. Mol Microbiol 510:2557–2568Google Scholar
  19. Koronakis V, Koronakis E, Hughes C (1989) Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J 8:595–605Google Scholar
  20. Kyhse-Anderson J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209Google Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  22. Letoffe S, Wandersman C (1992) Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B. J Bacteriol 174:4920–4927Google Scholar
  23. Letoffe S, Delepelaire P, Wandersman C (1991) Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions. J Bacteriol 173:2160–2166Google Scholar
  24. Ludwig A, Goebel W (1991) Genetic determinants of cytolytic toxins from gram-negative bacteria. In: Alouf JE, Freer JH (eds) Sourcebook of bacterial protein toxins. Academic Press, London, pp 117–146Google Scholar
  25. Mackman N, Nicaud J-M, Gray L, Holland IB (1985) Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001. Mol Gen Genet 201:282–288Google Scholar
  26. Mackman N, Baker K, Gray L, Haigh R, Nicaud J-M, Holland IB (1987) Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin. EMBO J 6:2835–2841Google Scholar
  27. Nicaud J-M, Mackman N, Gray L, Holland IB (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 204:331–335Google Scholar
  28. Olmsted JB (1986) Analysis of cytoskeletal structures using blotpurified monospecific antibodies. Methods Enzymol 134:467–472Google Scholar
  29. Oliver D-B (1987) Periplasm and protein secretion. In: Neidhardt, FC (ed) Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, DC, pp 56–69Google Scholar
  30. Pellett S, Boehm DF, Synder IS, Rowe G, Welch RA (1990) Characterization of monoclonal antibodies against the Escherichia coli hemolysin. Infect Immun 58:822–827Google Scholar
  31. Pugsley AP (1988) Protein secretion across the outer membrane of gram-negative bacteria. In: Das RC, Robbins PW (eds) Protein transfer and organelle biogenesis. Academic Press, San Diego, pp 607–652Google Scholar
  32. Wagner W, Vogel M, Goebel W (1983) Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol 154:200–210Google Scholar
  33. Wandersman C, Delepelaire P (1990) Tol C, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci USA 87:4776–4780Google Scholar
  34. Zagursky RJ, Baumeister K, Lomax N, Berman ML (1985) Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA. Gene Anal Technol 2:89–94Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • T. Jarchau
    • 1
  • T. Chakraborty
    • 1
  • F. Garcia
    • 1
  • W. Goebel
    • 1
  1. 1.Theodor-Boveri-Institut für BiowissenschaftenWürzburgGermany

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