1887

Abstract

A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from sp. CDF and expressed in . The enzyme gene is translated as a proform consisting of a 94 aa propeptide and a 283 aa mature protease domain. Phylogenetic analysis revealed that this enzyme belonged to the subtilisin family, but could not be grouped into any of its six known subfamilies. The mature protease CDF has an unusually high content of charged residues, which are mainly distributed on the enzyme surface. The recombinant proform of protease CDF formed inclusion bodies, but could be efficiently converted to the mature enzyme when the inclusion bodies were dissolved in alkaline buffers. The proform underwent a two-step maturation process, wherein the N-terminal part (85 residues) of the propeptide was autoprocessed intramolecularly, and the remaining 9-residue peptide was further processed intermolecularly. Protease CDF exhibited optimal proteolytic activity at 50–55 °C and pH 10.5–11.0. The enzyme was stable under high-pH conditions (pH 11.0–12.0), and NaCl could stabilize the enzyme at lower pH values. In addition, the enzyme was not dependent on calcium for either maturation or stability. By immunoblot analysis, protease CDF was found to be associated with spores, and could be extracted from the spores with 2 M KCl and alkaline buffers without damaging the coat layer, demonstrating that the protease CDF is located on the surface of the spore coat.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.031336-0
2009-11-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/11/3661.html?itemId=/content/journal/micro/10.1099/mic.0.031336-0&mimeType=html&fmt=ahah

References

  1. Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. ( 2004; ). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783–795.[CrossRef]
    [Google Scholar]
  2. Betzel, C., Pal, G. P. & Saenger, W. ( 1988; ). Three-dimensional structure of proteinase K at 0.15-nm resolution. Eur J Biochem 178, 155–171.[CrossRef]
    [Google Scholar]
  3. Boschwitz, H., Halvorson, H. O., Keynan, A. & Milner, Y. ( 1985; ). Trypsinlike enzymes from dormant and germinated spores of Bacillus cereus T and their possible involvement in germination. J Bacteriol 164, 302–309.
    [Google Scholar]
  4. Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B. & Power, S. ( 1988; ). The three-dimensional structure of Bacillus amyloliquefaciens subtilisin at 1.8 Å and an analysis of the structural consequences of peroxide inactivation. J Biol Chem 263, 7895–7906.
    [Google Scholar]
  5. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  6. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. ( 2003; ). Multiple sequence alignment with the clustal series of programs. Nucleic Acids Res 31, 3497–3500.[CrossRef]
    [Google Scholar]
  7. Comellas-Bigler, M., Maskos, K., Huber, R., Oyama, H., Oda, K. & Bode, W. ( 2004; ). 1.2 Å crystal structure of the serine carboxyl proteinase pro-kumamolisin; structure of an intact pro-subtilase. Structure 12, 1313–1323.[CrossRef]
    [Google Scholar]
  8. Gallagher, T., Gilliland, G., Wang, L. & Bryan, P. ( 1995; ). The prosegment-subtilisin BPN′ complex: crystal structure of a specific ‘foldase'. Structure 3, 907–914.[CrossRef]
    [Google Scholar]
  9. Gazenko, S. V., Reponen, T. A., Grinshpun, S. A. & Willeke, K. ( 1998; ). Analysis of airborne actinomycete spores with fluorogenic substrates. Appl Environ Microbiol 64, 4410–4415.
    [Google Scholar]
  10. Gros, P., Kalk, K. H. & Hol, W. G. ( 1991; ). Calcium binding to thermitase. Crystallographic studies of thermitase at 0, 5, and 100 mM calcium. J Biol Chem 266, 2953–2961.
    [Google Scholar]
  11. Hilbert, D. W. & Piggot, P. J. ( 2004; ). Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68, 234–262.[CrossRef]
    [Google Scholar]
  12. Horsburgh, G. J., Atrih, A. & Foster, S. J. ( 2003; ). Characterization of LytH, a differentiation-associated peptidoglycan hydrolase of Bacillus subtilis involved in endospore cortex maturation. J Bacteriol 185, 3813–3820.[CrossRef]
    [Google Scholar]
  13. Jacobs, M., Eliasson, M., Uhlen, M. & Flock, J. I. ( 1985; ). Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res 13, 8913–8926.[CrossRef]
    [Google Scholar]
  14. Kaneko, R., Koyama, N., Tsai, Y. C., Juang, R. Y., Yoda, K. & Yamasaki, M. ( 1989; ). Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain. J Bacteriol 171, 5232–5236.
    [Google Scholar]
  15. King, J. & Laemmli, U. K. ( 1971; ). Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol 62, 465–477.[CrossRef]
    [Google Scholar]
  16. Kleine, R. ( 1982; ). Properties of thermitase, a thermostable serine protease from Thermoactinomyces vulgaris. Acta Biol Med Ger 41, 89–102.
    [Google Scholar]
  17. Kojima, S., Minagawa, T. & Miura, K. ( 1997; ). The propeptide of subtilisin BPN′ as a temporary inhibitor and effect of an amino acid replacement on its inhibitory activity. FEBS Lett 411, 128–132.[CrossRef]
    [Google Scholar]
  18. Lacey, J. ( 1989; ). Thermoactinomycetes. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2573–2585. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore, MD: Lippincott, Williams & Wilkins.
  19. Lee, J. K., Kim, Y. O., Kim, H. K., Park, Y. S. & Oh, T. K. ( 1996; ). Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. Biosci Biotechnol Biochem 60, 840–846.[CrossRef]
    [Google Scholar]
  20. Liu, Y. G. & Whittier, R. F. ( 1995; ). Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674–681.[CrossRef]
    [Google Scholar]
  21. Makhatadze, G. I., Loladze, V. V., Ermolenko, D. N., Chen, X. & Thomas, S. T. ( 2003; ). Contribution of surface salt bridges to protein stability: guidelines for protein engineering. J Mol Biol 327, 1135–1148.[CrossRef]
    [Google Scholar]
  22. Markland, F. S. & Smith, E. L. ( 1967; ). Subtilisin BPN′. VII. Isolation of cyanogen bromide peptides and the complete amino acid sequence. J Biol Chem 242, 5198–5211.
    [Google Scholar]
  23. Masui, A., Fujiwara, N. & Imanaka, T. ( 1994; ). Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions. Appl Environ Microbiol 60, 3579–3584.
    [Google Scholar]
  24. McPhalen, C. A. & James, M. N. ( 1988; ). Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo. Biochemistry 27, 6582–6598.[CrossRef]
    [Google Scholar]
  25. Moriyama, R., Sugimoto, K., Zhang, H., Inoue, T. & Makino, S. ( 1998; ). A cysteine-dependent serine protease associated with the dormant spores of Bacillus cereus: purification of the protein and cloning of the corresponding gene. Biosci Biotechnol Biochem 62, 268–274.[CrossRef]
    [Google Scholar]
  26. Nilsson, M. & Renberg, I. ( 1990; ). Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history. Appl Environ Microbiol 56, 2025–2028.
    [Google Scholar]
  27. Orsini, M. & Romano-Spica, V. ( 2001; ). A microwave-based method for nucleic acid isolation from environmental samples. Lett Appl Microbiol 33, 17–20.[CrossRef]
    [Google Scholar]
  28. Pauwels, R., Devos, M., Callens, L. & van der Straeten, M. ( 1978; ). Respiratory hazards from proteolytic enzymes. Lancet 1, 669
    [Google Scholar]
  29. Pepys, J., Jenkins, P. A., Festenstein, G. N., Gregory, P. H., Lacey, M. E. & Skinner, F. A. ( 1963; ). Farmer's lung: thermophilic actinomycetes as a source of “farmer's lung hay” antigen. Lancet 2, 607–611.
    [Google Scholar]
  30. Ramarao, N. & Lereclus, D. ( 2005; ). The InhA1 metalloprotease allows spores of the B. cereus group to escape macrophages. Cell Microbiol 7, 1357–1364.[CrossRef]
    [Google Scholar]
  31. Roberts, R. C., Nelles, L. P., Treuhaft, M. W. & Marx, J. J. ( 1983; ). Isolation and possible relevance of Thermoactinomyces candidus proteinases in farmer's lung disease. Infect Immun 40, 553–562.
    [Google Scholar]
  32. Sarkar, G. & Sommer, S. S. ( 1990; ). The “megaprimer” method of site-directed mutagenesis. Biotechniques 8, 404–407.
    [Google Scholar]
  33. Schagger, H. & von Jagow, G. ( 1987; ). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379.[CrossRef]
    [Google Scholar]
  34. Schalinatus, E., Behnke, U. & Ruttloff, H. ( 1983a; ). Intracellular proteases of Thermoactinomyces vulgaris. 1. Occurrence of the proteases in the cell and dynamics of their formation during cultivation. Nahrung 27, 371–377.[CrossRef]
    [Google Scholar]
  35. Schalinatus, E., Ruttloff, H. & Behnke, U. ( 1983b; ). Intracellular proteases of Thermoactinomyces vulgaris. 2. Biochemical characterization of proteases in cell extracts. Nahrung 27, 379–386.[CrossRef]
    [Google Scholar]
  36. Siezen, R. J. & Leunissen, J. A. ( 1997; ). Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci 6, 501–523.
    [Google Scholar]
  37. Smith, C. A., Toogood, H. S., Baker, H. M., Daniel, R. M. & Baker, E. N. ( 1999; ). Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 Å resolution. J Mol Biol 294, 1027–1040.[CrossRef]
    [Google Scholar]
  38. Tanaka, S.-I., Matsumura, H., Koga, Y., Takano, K. & Kanaya, S. ( 2007; ). Four new crystal structures of Tk-subtilisin in unautoprocessed, autoprocessed and mature forms: insight into structural changes during maturation. J Mol Biol 372, 1055–1069.[CrossRef]
    [Google Scholar]
  39. Teplyakov, A. V., Kuranova, I. P., Harutyunyan, E. H., Vainshtein, B. K., Frommel, C., Hohne, W. E. & Wilson, K. S. ( 1990; ). Crystal structure of thermitase at 1.4 Å resolution. J Mol Biol 214, 261–279.[CrossRef]
    [Google Scholar]
  40. Tesone, C. & Torriani, A. ( 1975; ). Protease associated with spores of Bacillus cereus. J Bacteriol 124, 593–594.
    [Google Scholar]
  41. Tsuchiya, K., Nakamura, Y., Sakashita, H. & Kimura, T. ( 1992; ). Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682. Biosci Biotechnol Biochem 56, 246–250.[CrossRef]
    [Google Scholar]
  42. Tsuchiya, K., Ikeda, I., Tsuchiya, T. & Kimura, T. ( 1997; ). Cloning and expression of an intracellular alkaline protease gene from alkalophilic Thermoactinomyces sp. HS682. Biosci Biotechnol Biochem 61, 298–303.[CrossRef]
    [Google Scholar]
  43. Wong, S. L., Price, C. W., Goldfarb, D. S. & Doi, R. H. ( 1984; ). The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. Proc Natl Acad Sci U S A 81, 1184–1188.[CrossRef]
    [Google Scholar]
  44. Wu, J., Bian, Y., Tang, B., Chen, X., Shen, P. & Peng, Z. ( 2004; ). Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops. FEMS Microbiol Lett 230, 251–258.[CrossRef]
    [Google Scholar]
  45. Yabuta, Y., Takagi, H., Inouye, M. & Shinde, U. ( 2001; ). Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin. J Biol Chem 276, 44427–44434.[CrossRef]
    [Google Scholar]
  46. Yamagata, Y., Isshiki, K. & Ichishima, E. ( 1995; ). Subtilisin Sendai from alkalophilic Bacillus sp.: molecular and enzymatic properties of the enzyme and molecular cloning and characterization of the gene, aprS. Enzyme Microb Technol 17, 653–663.[CrossRef]
    [Google Scholar]
  47. Yike, I., Rand, T. & Dearborn, D. G. ( 2007; ). The role of fungal proteinases in pathophysiology of Stachybotrys chartarum. Mycopathologia 164, 171–181.[CrossRef]
    [Google Scholar]
  48. Yoon, J.-H., Kim, I.-G., Shin, Y.-K. & Park, Y.-H. ( 2005; ). Proposal of the genus Thermoactinomyces sensu stricto and three new genera, Laceyella, Thermoflavimicrobium and Seinonella, on the basis of phenotypic, phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 55, 395–400.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.031336-0
Loading
/content/journal/micro/10.1099/mic.0.031336-0
Loading

Data & Media loading...

Supplements

vol. , part 11, pp. 3661 - 3672

Supplementary figures [ PDF], 471kb, containing: (a) Nucleotide and deduced amino acid sequence of the protease CDF gene. (b) Location of the protease CDF gene in the genome of sp. CDF. Distribution of the charged amino acid residues of protease CDF. Unrooted phylogenetic tree of subtilase family A enzymes.



PDF
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