1887

Abstract

The gene encoding the calcium-dependent extracellular proteinase from ATCC 14581 was cloned in the vector pBR322 and expressed in HB101. The DNA sequence of the cloned 3·7 kb fragment revealed only one open reading frame consisting of1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation startsite (ATG). A possible promoter sequence (TAGACG for the −35 region and TATAAT for the −10 region) was found about 69 bp upstreamof the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide ‘pro’ sequence of 221 amino acids preceding the putative mature protein of 317 amino acidresidues. Amino acid sequence comparison revealed 84·5% homology between the mature protein and that of a thermolabile neutral protease from . It also shares 73% homology with the thermostable neutral proteases of and . The zinc-binding sites and the catalytic residues are completely conserved in allfour proteases. NprM has a temperature optimum of 58 °C, a pH optimum of between 6·4 and 7·2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.

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1993-01-01
2021-04-23
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References

  1. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513–1523
    [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heynecker H. L., Boyer H. W., Crosa J. H., Falkow S. 1977; Construction and characterization of new cloning vehicles. Gene 2:95–113
    [Google Scholar]
  3. Boyer H. W., Roulland-DUSSOIX D. 1969; A complementation analysis of the restriction and modification of DNA in E. coli. Journal of Molecular Biology 41:459–472
    [Google Scholar]
  4. Van Den Burg B., Enequist H. G., Van DEN HAAR M. E., Eijsink V. G.H., Stulp B. K., Venema G. 1991; A highly thermostable neutral protease from B. caldolyticus: cloning and expression of the gene in B. suhtilis and characterization of the gene product. Journal of Bacteriology 173:4107–4115
    [Google Scholar]
  5. Chaloupka J. 1969; Dual control of megateriopeptidase synthesis. Annales de l’Institut Pasteur 177:631–636
    [Google Scholar]
  6. Chaloupka J., Severin A. I., Sastry K. J., Kucerova H., Strnadova M. 1982; Differences in the regulation of exocellular proteinase synthesis during growth and sporogenesis of B. megaterium. Canadian Journal of Microbiology 28:1214–1218
    [Google Scholar]
  7. Colman P. M., Jansonius J. N., Matthews B. W. 1972; The structure of thermolysin: an electron density map at 2·3 Å resolution. Journal of Molecular Biology 70:701–724
    [Google Scholar]
  8. Debabov V. G. 1982; The industrial use of bacilli. In The Molecular Biology of the Bacilli I pp. 331–364, Edited by. D. A. Dubnau.; New York: Academic Press:
    [Google Scholar]
  9. Feder J., Garrett L. R., Wildi B. S. 1971; Studies on the role of calcium in thermolysin. Biochemistry 10:4552–4555
    [Google Scholar]
  10. Fuji M., Takagi M., Imanaka T., Aiba S. 1983; Molecular cloning of a thermostable neutral protease gene from B. stearo- thermophilus in a vector plasmid and its expression in B. stearo- thermophilus and B. subtilis. Journal of Bacteriology 154:831–837
    [Google Scholar]
  11. Hager P. W., Rabinowitz J. C. 1985; Translational specificity in B. subtilis. In The Molecular Biology of the Bacilli 11 pp. 1–32, Edited by. D. A. Dubnau.; New York: Academic Press.:
    [Google Scholar]
  12. Hawley D. K., Mcclure W. R. 1983; Compilation and analysis of E. coli promoter DNA sequences. Nucleic Acids Research 11:2237–2255
    [Google Scholar]
  13. Keay L., Feder J., Garrett L. R., Moseley M. H., Cirulis. N. 1971; B. megaterium neutral protease, a zinc-containing metallo-enzyme. Biochimica et Biophysica Acta 229:829–835
    [Google Scholar]
  14. Kester W. R., Matthews B. W. 1977a; Crystallographic study of the binding of dipeptide inhibitors to thermolysin: implications for the mechanism of catalysis. Biochemistry 16:2506–2516
    [Google Scholar]
  15. Kester W. R., Matthews B. W. 1977b; Comparison of the structure of carboxypeptidase A and thermolysin. Journal of Biological Chemistry 252:7704–7710
    [Google Scholar]
  16. Kreft J., Hughes C. 1982; Cloning vectors derived from plasmids and phage of Bacillus. Current Topics in Microbiology and Immunology 96:1–17
    [Google Scholar]
  17. Kreil G. 1981; Transfer of proteins across membranes. Annual Review of Biochemistry 50:317–348
    [Google Scholar]
  18. Kubo M., Murayama K., Seto K., Imanaka T. 1988; Highly thermostable neutral protease from B. stearothermophilus. Journal of Fermentation Technology 66:13–17
    [Google Scholar]
  19. Kubo M., Imanaka T. 1988; Cloning and nucleotide sequence of the highly thermostable neutral protease gene from B. stearo-thermophilus. Journal of General Microbiology 134:1883–1892
    [Google Scholar]
  20. Kushner S. R. 1978; An improved method for transformation of E. coli with ColEl-derived plasmids. In Genetic Engineering, pp. 17–23, Edited by. B. Boyer & S. Nicosia.; Amsterdam: Elsevier.:
    [Google Scholar]
  21. Latt S. A., Holmquist B., Vallee B. L. 1969; Thermolysin: a zinc metalloenzyme. Biochemical and Biophysical Research Communications 37:333–339
    [Google Scholar]
  22. Levy P. L., Paugburn M. K., Burstein Y., Ericsson L. H., , Neurath H., Walsh K. A. 1975; Evidence of homologous relationship between thermolysin and neutral proteases A of B. subtilis. Proceedings of the National Academy of Sciences of the United States of America 75:4341–4345
    [Google Scholar]
  23. Loshon C., Setlow P. 1982; B. megaterium spore protease: purification, radioimmunoassay, and analysis of antigen level and localization during growth, sporulation, and spore germination. Journal of Bacteriology 150:303–311
    [Google Scholar]
  24. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor; NY: Cold Spring Harbor Laboratory.:
    [Google Scholar]
  25. Matthews B., Jansonius J., Colman P., Schoenborn B., Dupourque D. 1972; Three-dimensional structure of thermolysin. Nature, London 238:37–41
    [Google Scholar]
  26. Messing J. 1983; New M13 vectors for cloning. Methods in Enzymology 101:20–37
    [Google Scholar]
  27. Millet J. 1968; Etude de la Mégatériopeptidase protéase exocellulaire de B. megaterium. I: Purification et propriétés générales. Bulletin de la Societé de Chimie Biologique 51:61–68
    [Google Scholar]
  28. Millet J., Archer R. 1969; Spécificité de la mégatériopeptidase: une amino-endopeptidase á caractére hydrophobe. European Journal of Biochemistry 9:456–462
    [Google Scholar]
  29. Millet J., Aubert J.-P. 1969; Étude de la mégatériopeptidase. protéase exocellulaire de B. megaterium III: Biosynthése et rôle physiologique. Annales de I‘lnstitut Pasteur 117:460–473
    [Google Scholar]
  30. Moran C. P., Jr Lang, Legrice N., Lee F. J., Stephens G., Sonenshine M., Pero .J A. L., Losick R. 1982; Nucleotide sequences that signal the initiation of transcription and translation in B. subtilis. Molecular and General Genetics 186:339–346
    [Google Scholar]
  31. Nicaud J. M., Mackmann G. I., Holland I. B. 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 Letters 204:231–235
    [Google Scholar]
  32. Nishiya Y., Imanaka T. 1990; Cloning and nucleotide sequence of the B. stearothermophilus neutral protease gene and its transcriptional activator gene. Journal of Bacteriology 172:4861–4869
    [Google Scholar]
  33. Pauptit R. A., Karlsson R., Picot D, Jenkins J. A., Niklaus-REIMER A.-S., Jansonius N. 1988; Crystal structure of neutral protease from B. cereus refined at 3·0 Å resolution and comparison with the homologous but more thermostable enzyme thermolysin. Journal of Molecular Biology 199:525–537
    [Google Scholar]
  34. Perlman D., Halvorson H. O. 1983; A putative signal peptidase recognition site and sequence in eucaryotic and procaryotic signal peptides. Journal of Molecular Biology 167:391–409
    [Google Scholar]
  35. Pugsley A. P. 1988; Protein secretion across the outer membrane of Gram-negative bacteria. In Protein Transfer and Organelle Biogenesis pp. 607–652, Edited by. R. C. Das & P. W. Robbins. Orlando.; Florida: Academic Press.:
    [Google Scholar]
  36. Rufo G. A., Sullivan B. J., Sloma A., Pero J. 1990; Isolation and characterization of a novel extracellular metalloprotease from B. subtilis. Journal of Bacteriology 72:1019–1023
    [Google Scholar]
  37. Saito H., Miura K. 1963; Preparation of trans-forming deoxy ribonucleic acid by phenol treatment. Biochimica et Biophysica Acta 72:619–629
    [Google Scholar]
  38. Sanger. F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-termination inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74:5463–5467
    [Google Scholar]
  39. Sidler W., Niederer E., Suter F., Zuber H. 1986; The primary structure of B. cereus neutral protease and comparison with thermolysin and B. subtilis neutral proteinase. Biological Chemistry Hoppe-Seyler 367:643–657
    [Google Scholar]
  40. Skerman V. B.D., Mcgowan V., Sneath P. H.A. 1980; Approved lists of bacterial names. International Journal of Systematic Bacteriology 30:225–420
    [Google Scholar]
  41. Sloma A., Rudolph C. F., Rufo G. A., Jr Sullivan, Theriault B. J., K. A. ALLY D., Pero J. 1990; Gene encoding a novel extracellular metalloprotease in B. subtilis. Journal of Bacteriology 172:1024–1029
    [Google Scholar]
  42. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:503–517
    [Google Scholar]
  43. Stahl M. F., Ferrari E. 1984; Replacement of the B. subtilis subtilisin structural gene with an in vitro derived deletion mutation. Journal of Bacteriology 158:411–418
    [Google Scholar]
  44. Staufer C. E. 1971; The effect of pH on thermolysin activity. Archives of Biochemistry 147:568–570
    [Google Scholar]
  45. Takagi M., Imanaka T, Aiba A. 1985; Nucleotide sequence and promoter region for the neutral protease gene from B. stearo- thermophilus. Journal of Bacteriology 163:824–831
    [Google Scholar]
  46. Tinoco I., Jr. Borer, Dengler P. N., Fevin B., Uhlenbeck M. D., Crothers O. C., M. D., Graller J. 1973; Improved estimation of secondary structure in ribonucleic acids. Nature New Biology 246:40–41
    [Google Scholar]
  47. Titani K., Hermodson M., Ericsson L., Walsh. K., Neurath H. 1972; Amino acid sequence of thermolysin. Nature, London 238:35–37
    [Google Scholar]
  48. Tran F., Wu X.-C., Wong S.-F. 1991; Cloning and expression of a novel protease gene encoding an extracellular neutral protease from B. subtilis. Journal of Bacteriology 173:6364–6372
    [Google Scholar]
  49. Vasantha N., Thompson F. D., Rhodes C., Banner C., Nagle. J., Filpula D. 1984; Genes for alkaline protease and neutral protease from B. amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. Journal of Bacteriology 159:811–819
    [Google Scholar]
  50. Wong S.-L., Price C. W., Goldfarb D. S., Doi. R. H. 1984; The subtilisin E gene of B. subtilis is transcribed from a sigma37 promoter in vivo. Proceedings of the National Academy of Sciences of the United States of America 81:1184–1188
    [Google Scholar]
  51. Wu X.-C., Nathoo S., Pang A. S.-H., Carne. T., Wong S.-L. 1990; Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from B. subtilis. Journal of Biological Chemistry 265:6845–6850
    [Google Scholar]
  52. Yang M. Y., Ferrari E., Henner D. J. 1984; Cloning of the neutral protease gene of B. subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. Journal of Bacteriology 160:15–21
    [Google Scholar]
  53. Yoshimoto T., Oyama H., Takeshita T., Higashi H., Lin XU. S., Tsuru D. 1990; Nucleotide sequence of the neutral protease gene from B. subtilis var. amylosacchariticus. Journal of Fermentation and Bioengineering 70:370–375
    [Google Scholar]
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