Molecular and General Genetics MGG

, Volume 233, Issue 1–2, pp 42–48 | Cite as

Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by theEscherichia coli hemolysin transport system

  • Christian Hanke
  • Jürgen Hess
  • Günter Schumacher
  • Werner Goebel
Article

Summary

A fusion gene (ces-hlyAs) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyAs) ofEscherichia coli hemolysin (H1yA) to the ces gene for a cholesterol esterase/lipase (CE) from aPseudomonas species. Part (about 30 %) of the expressed fusion protein CE-H1yAs was secreted inE. coli carryinghlyB andhlyD genes. Following the insertion between the reporter gene andhlyAs of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-H1yAs and CE-HlyAs) were shown to be cleaved by OmpT between the two parts during H1yB/H1yD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increasedompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.

Key words

Secretion Recombinant DNA Hemolysin HlyB/H1yD complementation OmpT protease 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bagdasarian M, Bagdasarian MM, Coleman S, Timmis KN (1979) New Vector Plasmids for Gene Cloning in Pseudomonas. In: Timmis KN, Pilhler A (eds) Plasmids of environmental and commercial importance. Elsevier north Holland, Amsterdam, pp 411–422Google Scholar
  2. Beckwith J, Strauch KL (1988) AnEscherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci USA 85:1567–1580Google Scholar
  3. Bieker KL, Phillips GJ, Silhavy TJ (1990) Thesec andpol, genes ofEscherichia coli. J Bioenerg Biomembr 22:291–310Google Scholar
  4. Blight MA, Holland IB (1990) Structure and function of haemolysin B, P-glycoprotein and other members of a novel family of membrane translocators. Mol Microbiol 4:873–80Google Scholar
  5. Brundage L, Hendrick JP, Schiebel E, Driessen AJM, Wickner W (1990) The purifiedE. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA dependent precursor protein translocation. Cell 62:649–657Google Scholar
  6. Erhart CF, Lundrigan M, Pickett CL, Pierce RP (1979)Escherichia coli K-12 mutants that lack major outer membrane protein a. FEMS Microbiol Rev 6:277–280Google Scholar
  7. 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
  8. Felmlee T, Pellett S, Lee E-Y, Welch R (1985)Escherichia coli hemolysin is released extracellulary without cleavage of a signal peptide. J Bacteriol 163:88–93Google Scholar
  9. 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
  10. Gerlach JJ, Endicott JA, Juranka PF, Henderson G, Sarangi F, Deuchars KL, Ling V (1986) Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance. Nature 324:485–489Google Scholar
  11. Goebel W, Hedgpeth J (1982) Cloning and functional characterization of the plasmid-encoded hemolysin determinant of E. coli. J Bacteriol 151:1290–1298Google Scholar
  12. Goff SA, Goldberg AL (1985) Production of abnormal proteins inE. coli stimulates transcription oflon and other heat shock proteins. Cell 41:587–595Google Scholar
  13. Gray L, Baker K, Kenny B, Mackman N, Haigh R, Holland IB (1989) Protein targeting. J Cell Sci [Suppl] 11:45–57Google Scholar
  14. Grodberg J, Dunn JJ (1988)OmpT encodes theEscherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol 170:1245–1253Google Scholar
  15. Grodberg J, Dunn JJ (1989) Comparison ofEscherichia coli K12 outer membrane protease ompT andSalmonella typhimurium E protein. J Bacteriol 171:2903–2905Google Scholar
  16. Gros P, Croop J, Hosuman D (1986) Mammalian multidrug resistance gene: complete cDNA indicates strong homology to bacterial transport proteins. Cell 47:371–380Google Scholar
  17. Hackett J, Reeves P (1983) Primary structure of thetolC gene that codes for an outer membrane protein ofEscherichia coli K12. Nucleic Acids Res 11:6487–6495Google Scholar
  18. Härtlein M, Schießl S, Wagner W, Rdest U, Kreft J, Goebel W (1983) Transport of hemolysin byEscherichia coli. J Cell Biochem 22:87–97Google Scholar
  19. Hellebust H, Murby M, Abrahamsen L, Uhlen M, Enfors S-O (1989) Different approaches to stabilize a recombinant fusion protein. Biotechnology 7:165–168Google Scholar
  20. Hemmingsen SM, Woolford C, Saskia M, van der Vies SM, Tilly K, Dennis DT, Georgopoulos CP, Hendriks RW, Ellis RJ (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333: 330–334Google Scholar
  21. Hess J, Gentschev I, Goebel W, Jarchau T (1990) Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins. Mol Gen Genet 224:201–208Google Scholar
  22. Holland B, Kenny B, Blight M (1990) Haemolysin secretion fromE. coli. Biochimie 72:131–141Google Scholar
  23. Koronakis V, Koronakis E, Hughes C (1989) Isolation and analysis of the C-terminal signal directing export ofEscherichia coli haemolysin protein across both bacterial membranes. EMBO J 8:595–605Google Scholar
  24. Kusukawa N, Yura T, Uegushi C, Akiyama Y, Ito K (1990) Effects of mutations in heat shock genes groES and groEL on protein export inEscherichia coli. EMBO J 8:3517–3521Google Scholar
  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  26. Leong J, Neilands JB (1976) Mechanisms of siderophore iron transport in enteric bacteria. J Bacteriol 126:823–830Google Scholar
  27. Lill R, Dowhan W, Wickner W (1989) The ATPase activity of SecA is regulated by acidic phospholipids, SecY and the leader and mature domains of precursor proteins. Cell 60:271–280Google Scholar
  28. Lill R, Cunningham L, Brundage L, Ito K, Oliver D, Wickner W (1990) The SecA protein hydrolyzes ATP and is an essential component of protein translocation ATPase. EMBO J 8:961–966Google Scholar
  29. Lipinska B, Zylicz M, Georgopoulos C (1990) The htrA (degP) protein, essential forEscherichia coli survival at high temperatures, is an endopeptidase. J Bacteriol 172:1791–1797Google Scholar
  30. Ludwig A, Vogel M, Goebel W (1987) Mutations affecting activity and transport of haemolysin inE. coli. Mol Gen Genet 206:238–245Google Scholar
  31. Mackman N, Baker K, Gray L, Haigh R, Nicaud J-M, Holland IB (1987) Release of a chimeric protein into the medium fromEscherichia coli using the C-terminal secretion signal of haemolysin. EMBO J 6:2835–2841Google Scholar
  32. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  33. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78Google Scholar
  34. Messing J, Crea R, Seeburg PN (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–313Google Scholar
  35. Nicaud J-M, Mackman N, Gray L, Holland IB (1986) The C-terminal, 23kD peptide ofEscherichia coli haemolysin determinant 2001 contains all the information necessary for its secretion by the haemolysin (Hly) export machinery. FEBS Lett 204:331–335Google Scholar
  36. Oliver DB, Beckwith J (1982) Regulation of a membrane component required for protein secretion inEscherichia coli. Cell 30:311–319Google Scholar
  37. Pugsley AP (1988) Protein secretion across the outer membrane of gram-negative bacteria. In: Das RA, Robbins PW (eds) Protein transfer and organelle biogenesis. Academic Press, New York, pp 606–652Google Scholar
  38. Pugsley AP, Schwartz M (1985) Export and secretion of proteins by bacteria. FEMS Microbiol Rev 32:3–38Google Scholar
  39. Raina S, Georgopoulos C (1990) A newEscherichia coli heat shock gene,htrC, whose product is essential for viability only at high temperatures. J Bacteriol 172:3417–3426Google Scholar
  40. Randall LL, Hardy SJ (1986) Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein ofE. coli. Cell 46:921–928Google Scholar
  41. Schatz PJ, Riggs PD, Fath AJ, Beckwith J (1989) The secE gene encodes an integral membrane protein required for protein export inEscherichia coli. Genes Dev 3:1035–1044Google Scholar
  42. Schumacher G (1986) Creatinase-encoding plasmid construction and creatinase production byEscherichia coli orPseudomonas putida transformed by the plasmid. European Patent Office, Patent No. EPA 187138 A2Google Scholar
  43. Sedgwick B (1989) In vitro proteolytic cleavage of theEscherichia coli Ada protein by theompT gene product. J Bacteriol 171:2249–2251Google Scholar
  44. Shiba K, Ito K, Yura T, Cerretti DP (1984) A defined mutation in the protein export within the spc ribosomal protein operon ofEscherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J 3:631–635Google Scholar
  45. Sugimura K, Higashi N (1988) A novel outer-membrane-associated protease inEscherichia coli. J Bacteriol 170:3650–3654Google Scholar
  46. Sugimura K, Nishihashi T (1988) Purification, characterization and primary structure ofEscherichia coli protease VII with specificity for paired basic residues. J Bacterol 170:5625–5632Google Scholar
  47. 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 76:4350–4354Google Scholar
  48. Wagner W, Vogel M, Goebel W (1983) Transport of haemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol 154:200–210Google Scholar
  49. Wandersman C, Delepelaire P (1990) TolC, anEscherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci USA 87:4776–4780Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Christian Hanke
    • 2
  • Jürgen Hess
    • 1
  • Günter Schumacher
    • 2
  • Werner Goebel
    • 1
  1. 1.Institut für Genetik und MikrobiologieUniversity of WürzburgWürzburgGermany
  2. 2.Abteilung für GenetikPenzbergGermany

Personalised recommendations