1887

Abstract

The α-amylase gene, , was expressed under the regulated control of , the levansucrase leader region. The gene fusion including the complete coding sequence with the signal peptide sequence was integrated into the chromosome of a (Hy) strain deleted of the DNA fragment. In this genetic context, α-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step ( = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of α-amylase occurring within the cell wall environment was substantiated by folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range ( = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied by the integrity of the cell wail. The of the α-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-143-10-3295
1997-10-01
2024-12-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/143/10/mic-143-10-3295.html?itemId=/content/journal/micro/10.1099/00221287-143-10-3295&mimeType=html&fmt=ahah

References

  1. Archibald A.R. 1989; The bacillus cell envelope. In Bacillus pp. 217–254 Edited by Harwood C. R. New York: Plenum;
    [Google Scholar]
  2. Bauw G., Van Damme J., Puype M., Vandekerckhove J., Gesser B., Ratz G.P., Lauridsen J.B., Celis J.E. 1989; Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels. Proc Natl Acad Sci USA 86:7701–7705
    [Google Scholar]
  3. Beveridge T.J. 1995; The periplasmic space and the periplasm in gram-positive and gram-negative bacteria. asm. News 61:125–130
    [Google Scholar]
  4. Chambert R., Petit-Glatron M.-F. 1984; Hyperproduction of exocellular levansucrase by bacillus subtilis: Examination of the phenotype of a sacuh strain. J Gen Microbiol 130:3143–3152
    [Google Scholar]
  5. Chambert R., Petit-Glatron M.-F. 1988; Secretion mechanism of bacillus subtilis levansucrase: Characterization of the second step. J Gen Microbiol 134:1205–1214
    [Google Scholar]
  6. Chambert R., Petit-Glatron M.-F. 1989; Study of the effect of organic solvents on the synthesis of levan and the hydrolysis of sucrose by bacillus subtilis levansucrase. Carbohydr Res 191:117–123
    [Google Scholar]
  7. Chambert R., Haddaoui E.A., Petit-Glatron M.-F. 1995; Bacillus subtilis. Microbiology 141:997–1005
    [Google Scholar]
  8. Ferrari E., Nguyen A., Lang D., Hoch J. 1983; Construction and properties of an integrable plasmid for b. Subtilis. I Bacteriol 154:1513–1515
    [Google Scholar]
  9. Graham L.L., Beveridge T.J. 1994; Structural differentiation of the bacillus subtilis 168 cell wall. J Bacteriol 176:1413–1421
    [Google Scholar]
  10. Guérout-Fleury A.M., Shazand K., Frandsen N., Stragier P. 1995; Antibiotic-resistance cassettes for bacillus subtilis. Gene 167:335–336
    [Google Scholar]
  11. Haddaoui E.A., Chambert R., Petit-Glatron M.-F. 1995; Characterization of a new cell-bound α-amylase in bacillus subtilis 168 marburg only immunologically related to the exocellular α-amylase. J Bacteriol 177:5148–5150
    [Google Scholar]
  12. Koch A. 1995; Gram-positive rod-shaped organisms: Bacillus subtilis. In Bacterial Growth and Form pp. 219–249 New York: Chapman & Hall;
    [Google Scholar]
  13. Lepesant J.A., Kunst F., Pascal M., Lepesant-Kejzlarova J., Steinmetz M., Dedonder R. 1976; Specific and pleiotropic regulatory mechanism in the sucrose system of bacillus subtilis. In Microbiology –1976 pp. 58–69 Edited by Schlessinger D. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  14. Mäntsälä P., Zalkin H. 1979; Membrane-bound and soluble extracellular α-amylase from bacillus subtilis. J Biol Chem 254:8540–8547
    [Google Scholar]
  15. Merchante R., Pooley H.M., Karamata D. 1995; A periplasm in bacillus subtilis. J Bacteriol 177:6176–6183
    [Google Scholar]
  16. Nagata Y., Yamaguchi K., Maruo B. 1974; Genetic and biochemical studies on cell-bound α-amylase in bacillus subtilis marburg. J Bacteriol 119:425–430
    [Google Scholar]
  17. Nakamura K., Nishiguchi M., Honda K., Yamane K. 1994; The bacillus subtilis srp54 homologue, ffh, has an intrinsic gtpase activity and forms a ribonucleoprotein complex with small cytoplasmic rna in vivo. Biochem Biophys Res Commun 199:1394–1399
    [Google Scholar]
  18. Noltmann E.A. 1966; Phosphoglucose isomerase. Methods Enzymol 9:557–568
    [Google Scholar]
  19. Ogura A., Kakeshita H., Takamatsu H., Nakamura K., Yamane K. 1996; The effect of srb, a homologue of the mammalian srp receptor alpha-subunit, on bacillus subtilis growth and protein translocation. Gene 172:17–24
    [Google Scholar]
  20. Petit-Glatron M.-F., Chambert R. 1992; Peptide carrier potentiality of bacillus subtilis levansucrase. J Gen Microbiol 138:1089–1095
    [Google Scholar]
  21. Petit-Glatron M.-F., Benyahia F., Chambert R. 1987; Secretion of bacillus subtilis levansucrase: A possible two step mechanism. Eur J Biochem 163:379–387
    [Google Scholar]
  22. Petit-Glatron M.-F., Grajcar L., Munz A., Chambert R. 1993; The contribution of the cell wall to a transmembrane calcium gradient could play a key role in bacillus subtilis protein secretion. Mol Microbiol 9:1097–1106
    [Google Scholar]
  23. Pooley H., Merchante R., Karamata D. 1996; Overall protein content and induced enzyme components of the periplasm of bacillus subtilis. Microb Drug Resist 2:9–15
    [Google Scholar]
  24. Sakakibara Y., Tsutsumi K., Nakamura K., Yamane K. 1993; Structural requirements of bacillus subtilis α-amylase signal peptide for efficient processing: In vivo pulse-chase experiments with mutant signal peptides. J Bacteriol 175:4203–4212
    [Google Scholar]
  25. Sasamoto H., Nakazawa K., Tsutsumi K., Takase K., Yamane K. 1989; Signal peptide of bacillus subtilis α-amylase. J Biochem 106:376–382
    [Google Scholar]
  26. Simonen M., Palva I. 1993; Protein secretion in bacillus species. Microbiol Rev 57:109–137
    [Google Scholar]
  27. Steinmetz M., Le Coq D., Aymerich S., Gonzy-Treboul G., Gay P. 1985; The dna sequence of the gene for the secreted bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet 200:220–228
    [Google Scholar]
  28. Takase K., Mizuno H., Yamane K. 1988; Nh2-terminal processing of bacillus subtilis a-amylase. J Biol Chem 263:11548–11553
    [Google Scholar]
  29. Weickert M.J., Chambliss G.H. 1989; Genetic analysis of the promoter region of the bacillus subtilis α-amylase gene. J Bacteriol 111:3656–3666
    [Google Scholar]
  30. Yang M., Galizzi A., Henner D. 1983; Nucleotide sequence of the amylase gene from. Nucleic Acids Res 11:237–249
    [Google Scholar]
/content/journal/micro/10.1099/00221287-143-10-3295
Loading
/content/journal/micro/10.1099/00221287-143-10-3295
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error