Molecular and General Genetics MGG

, Volume 204, Issue 2, pp 289–295 | Cite as

Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila

  • S. Peter Howard
  • J. Thomas Buckley
Article

Summary

The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8+. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8+ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.

Key words

Extracellular Toxin Cloning Signal Localization 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523Google Scholar
  2. De Bruijn FJ, Lupski JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids-a review. Gene 27:131–149Google Scholar
  3. De Graaf FK, Klassen-Boor P (1977) Purification and characterization of a complex between cloacin and its immunity protein isolated from Enterobactercloacae (Clo DF13) Eur J Biochem 73:107–114Google Scholar
  4. Felmlee T, Pellet S, Lee E-Y, Welch RA (1985) Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J Bacteriol 163:88–93Google Scholar
  5. Foster JW, Kinney DM (1985) ADP-ribosylating microbial toxins. Crit Rev in Microbiol 11:273–298Google Scholar
  6. Gennaro ML, Greenway PJ, Broadbent DA (1982) The expression of biologically active cholera toxin in Escherichia coli. Nucleic Acids Res 10:4883–4890Google Scholar
  7. Godson GN, Vapnek D (1973) A simple method of preparing large amounts of phi-x174 RF1 super coiled DNA. Biochim Biophys Acta 299:516–520Google Scholar
  8. Goldberg SL, Murphy JR (1984) Molecular cloning of the hemolysin determinant from Vibrio cholera El Tor. J Bacteriol 160:239–244Google Scholar
  9. Gray GL, Smith DH, Baldridge JS, Harkins RN, Vasil ML, Chen EY, Heyneker HL (1984) Cloning, nucleotide sequence and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 81:2645–2649Google Scholar
  10. Guzman-Verduzco LM, Fonseca R, Kupersztoch-Portnoy, YM (1983) Thermoactivation of a periplasmic heat-stable enterotoxin of Escherichia coli. J Bacteriol 154:146–151Google Scholar
  11. Hirst TR, Randall LL, Hardy SJS (1984) Cellular location of heatlabile enterotoxin in Escherichia coli. J Bacteriol 157:637–642Google Scholar
  12. Howard SP, Buckley JT (1982) Membrane glycoprotein receptor and hole-forming properties of a cytolytic protein toxin. Biochemistry 21:1662–1667Google Scholar
  13. Howard SP, Buckley JT (1983) Intracellular accumulation of extracellular proteins by pleiotropic export mutants of Aeromonas hydrophila. J Bacteriol 154:413–418Google Scholar
  14. Howard SP, Buckley JT (1985a) Activation of the hole forming toxin aerolysin by extracellular processing. J Bacteriol 163:336–340Google Scholar
  15. Howard SP, Buckley JT (1985b) Protein export by a gram negative bacterium: production of aerolysin by Aeromonas hydrophila. J Bacteriol 161:1118–1124Google Scholar
  16. Jakes KS, Model P (1979) Mechanism of export of colicin E1 and colicin E3. J Bacteriol 138:770–778Google Scholar
  17. Knauf VC, Nester EW (1982) Wide host range cloning vectors: a cosmid clone bank of an agrobacterium Ti plasmid. Plasmid 8:45–54Google Scholar
  18. Lewin B (1983) Genes. Wiley, New York, pp 219–257Google Scholar
  19. Lory S, Tai PC, Davis BD (1983) Mechanism of protein excretion by gram-negative bacteria: Pseudomonas aeruginosa exotoxin A. J Bacteriol 156:695–702Google Scholar
  20. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NYGoogle Scholar
  21. McClure WR (1985) Mechanism and control of transcription initiation in procarvotes. Annu Rev Biochem 54:171–204Google Scholar
  22. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–77Google Scholar
  23. Meyer TF, Mlawer N, So M (1982) Pilus expression in Niesseria gonorrhoeae involves chromasomal rearrangement. Cell 30:45–52Google Scholar
  24. Mock M, Schwartz M (1978) Mechanism of colicin E3 production in strains harboring wild-type or mutant plasmids. J Bacteriol 136:700–707Google Scholar
  25. Morlon J, Lloubes R, Varenne S, Chartier M, Lazdunski C (1983) Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate. J Mol Biol 170:271–285Google Scholar
  26. Neville DM (1971) Molecular weight determination of proteindodecyl sulphate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem 246:6328–6334Google Scholar
  27. Nishibuchi M, Kaper J (1985) Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahemolyticus. J Bacteriol 162:558–564Google Scholar
  28. Puhler A, Timmis KN (1984) Advanced molecular genetics. Springer, Berlin Heidelberg New YorkGoogle Scholar
  29. Sanger F, Nicklen S, Coulson AR (1979) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  30. Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature (Lond) 254:34–38Google Scholar
  31. Springer W, Goebel W (1980) Synthesis and secretion of hemolysin by Escherichia coli. J Bacteriol 144:53–59Google Scholar
  32. Taniguchi H, Ohta H, Ogawa M, Mizuguchi Y (1985) Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes. J Bacteriol 162:510–515Google Scholar
  33. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:5350–5354Google Scholar
  34. Vlasuk GP, Inouye S, Ito H, Itakura K, Inouye M (1983) Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J Biol Chem 258:7141–7148Google Scholar
  35. Von Heijne G (1984) How signal sequences maintain cleavage specificity. J Mol Biol 173:243–251Google Scholar
  36. 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
  37. Willis RC, Morris RG, Cirakoglu C, Schellenberg GD, Gerber NH, Furlong CE (1974) Preparation of the periplasmic binding proteins from Salmonella typhimurium and Escherichia coli. Arch Biochem Biophys 161:64–75Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • S. Peter Howard
    • 1
  • J. Thomas Buckley
    • 1
  1. 1.Department of Biochemistry and MicrobiologyUniversity of VictoriaVictoriaCanada

Personalised recommendations