1887

Abstract

Substitutions of an aspartate or an arginine residue for the glycine residue at position 366 of the mature part of levansucrase were obtained by mutagenesis. Quantitative estimation from immunoblot analysis showed that the two transient membrane forms of the modified proteins were present in the membrane at the same level as that of the wild-type protein. The proteolytic processing, which was previously shown to be the first step of the levansucrase secretion process, was not affected in these modified proteins. Results from pulse-chase experiments showed that the half-times for secretion of the modified levansucrases into the culture medium were nearly the same as that of the wild-type protein, but the amount of the modified proteins secreted was significantly reduced. Purified samples of the modified enzymes were subtilisin insensitive and possessed enzyme activities very similar to those of the wild-type enzyme. The results suggest that the 366 site probably belongs to a functional domain of the protein which could play an important role in the second step of the levansucrase secretion process.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-134-12-3259
1988-12-01
2024-12-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/134/12/mic-134-12-3259.html?itemId=/content/journal/micro/10.1099/00221287-134-12-3259&mimeType=html&fmt=ahah

References

  1. Aymerich S., Gonzy-Trbboul G.A, Steinmetz M. 1986; 5´-Noncoding region sacR is the target of all identified regulations affecting the levansucrase gene in Bacillus subtilis . Journal of Bacteriology 166:993–998
    [Google Scholar]
  2. Chambbrt R., Petit-Glatron M. F. 1984; Hyperproduction of exocellular levansucrase by Bacillus subtilis : examination of the phenotype of a sacUh strain. Journal of General Microbiology 130:3143–3152
    [Google Scholar]
  3. Chambbrt R., Petit-Glatron M. F. 1988; Secretion mechanism of Bacillus subtilis levansucrase : characterization of the second step. Journal of General Microbiology 134:1205–1214
    [Google Scholar]
  4. Chambert R., Trbboul G., Dhdonder R. 1974; Kinetic studies of levansucrase of Bacillus subtilis . European Journal of Biochemistry 41:285–300
    [Google Scholar]
  5. Coleman G., Elliott W. H. 1962; Studies on aamylase formation by Bacillus subtilis . Biochemical Journal 83:256–263
    [Google Scholar]
  6. Dente L., Sollazzo M., Baldari C., Cesarbni G., Cortese R. 1985; The pEMBL family of singlestranded vectors. In DNA Cloning, a Practical Approach pp. 101–107 Glover D. M. Edited by Oxford & Washington DC: IRL Press;
    [Google Scholar]
  7. Eilbrs M., Schatz G. 1986; Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature; London: 322228–232
    [Google Scholar]
  8. Fbrbnci T., Silhavy T. J. 1987; Sequence information required for protein translocation from the cytoplasm. Journal of Bacteriology 169:5339–5342
    [Google Scholar]
  9. Fitts R., Reuveny Z., Van amsterdam J., Mul-Holland J., Botstein D. 1987; substitution of tyrosine for either cysteine in /Mactamase prevents release from the membrane during secretion. Proceedings of the National Academy of Sciences of the United States of America 84:8540–8543
    [Google Scholar]
  10. Freudl R., Schwarz H., Stierhof Y. D., Gamon K., Hindennach I., Henning U. 1986; An outer membrane protein (OmpA) of Escherichia coli K12 undergoes a conformational change during export. Journal of Biological Chemistry 261:11355–11361
    [Google Scholar]
  11. Himeno T., Imanaka T., Aiba S. 1986; Protein secretion in Bacillus subtilis as influenced by the combination of signal sequence and the following mature portion. FEMS Microbiology Letters 35:17–21
    [Google Scholar]
  12. Kunst F., Pascal M., Lbpbsant-Kejzlarova J., Lepersant J. A., Billault A., Dedondbr R. 1974; Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168. Biochimie 56:1481–1489
    [Google Scholar]
  13. Lebrun E. 1980; Structure tridimensionnelle de la levane saccharose de Bacillus subtilis a 3,8 Å de resolution. These de Doctoral, Universite Paris VI
    [Google Scholar]
  14. Le Coq D., Ratet P., Steinmetz M., Gay P. 1984; A genetic approach to levansucrase secretion in Bacillus Subllius . In Genetics and Btotechnology of Bacilli pp. 141–152 Ganesan J., Hoch A. Edited by Academic Press; New York:
    [Google Scholar]
  15. Levin D., Joyet P., Louvbncourt L., Aymerich S., Le reverend B., Steinmetz M., Heslot H. 1985; Vecteurs d’expression de I’aαamylase dans Bacillus scrbtilis, a ches obtenues et procede preparation d α-amylase.Brevet no. 2582316. Institut National de la Propriete Industrielle; Paris:
    [Google Scholar]
  16. Maher P. A., Singer S. J. 1986; Disulfide bonds and the translocation of proteins across membranes. Proceedings of the National Academy of Sciences of the United States of America 83:9001–9005
    [Google Scholar]
  17. Park S., Liu G., Topping T. B., Covbr W. H. 1988; Modulation of folding pathways of exported proteins by the leader sequence. Science 239:1033–1035
    [Google Scholar]
  18. Petit-Glatron M. F., Chambert R. 1981; Levansucrase of Bacillus subtilis: conclusive evidence that its production and export are unrelated to fatty-acid synthesis but modulated by membranemodifying agents. European Journal of Biochemistry 119:603–611
    [Google Scholar]
  19. Petit-Glatron M. F., Benyawa F., Chambert R. 1987; Secretion of Bacillus subtilis levansucrase: a possible two-step mechanism. European Journal of Biochemistry 163:379–387
    [Google Scholar]
  20. Randall L. L., Hardy S. J. S. 1986; Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in Escherichia coli . Cell 46:921–928
    [Google Scholar]
  21. Sanger F., Coulson A. R., Barrbll B. G., Smith A. J. H., Roe B. A. 1980; Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. Journal of Molecular Biology 143:161–178
    [Google Scholar]
  22. Schultz S. C., Dalbadie-Mcfarland G., Nbitzel J. J., Richards J. H. 1987; Stability of wild type and mutant RTEM-1 β-lactamases: effect of the disulfide bond. Proteins: Structure, Function and Genetics 2:290–297
    [Google Scholar]
  23. Shimotsu H., Henner D. J. 1986; Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. Journal of Bacteriology 168:380–388
    [Google Scholar]
  24. Steinmetz M., Le Coq D., Ben Djemia H., Gay P. 1983; Analyse g6netique de sacB, gene de structure d’une enzyme secrete, la levane-saccharase de Bacillus subtilis . Molecular and General Genetics 191:138–144
    [Google Scholar]
  25. Steinmetz M., Le Coq D., Aymerich S., Gonzy-Trbboul G., Gay P. 1985; The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Molecular and General Genetics 200:220–228
    [Google Scholar]
  26. Vieira J., Mbssing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
    [Google Scholar]
  27. Wickner W. T., Lodish H. F. 1985; Multiple mechanisms of protein insertion into and across membranes. Science 230:400–407
    [Google Scholar]
  28. Yutani K., Ogasahara K., Tsujita T., Sugino Y. 1987; Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase a subunit. Proceedings of the National Academy of Sciences of the United States of America 84:4441–4444
    [Google Scholar]
  29. Zoller M. J., Smith M. 1984; Oligonucleotide directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA 3:479–488
    [Google Scholar]
/content/journal/micro/10.1099/00221287-134-12-3259
Loading
/content/journal/micro/10.1099/00221287-134-12-3259
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