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Overproduction of Encapsulated Insecticidal Crystal Proteins in a Bacillus thuringiensis spoOA Mutant

Bio/Technology volume 13, pages 67–71 (1995)Cite this article

Abstract

The spoOA gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spoOA mutant of B. subtilis. In this paper we describethe construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spoOA gene of Bt was cloned and identified by its ability to transform a B. subtilis spoOA mutant to prototrophy. Its nucleotidesequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a dis rupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spoOA mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulatedtoxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spoOA mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

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References

  1. Rigby, S. 1991. Bt in crop protection, PJB Publications, Richmond, UK.

    Google Scholar 

  2. Feitelson, J.S., Payne, J., Kirn, L. . 1992. Bacillus thuringiensis: insects and beyond. Bio/Technology 10: 271–275.

    Google Scholar 

  3. Lambert, B., Peferoen, M. 1992. Insecticidal promise of Bacillus thuringiensis. BioScience 42: 112–122.

    Article  Google Scholar 

  4. van Frankenhuyzen, K. 1993. The challenge of Bacillus thuringiensis, 1–35. In: Bacillus thuringiensis, an Environmental Biopesticide: Theory and Practice. Entwistle, P. F, Cory, J. S., Bailey, M. J. and Higgs, S. (Eds). John Wiley & Sons Ltd, Chichester, UK.

    Google Scholar 

  5. Gelernter, W.D. 1990. Targeting insecticide-resistant market: new develop ments in microbial based products, 105–117. In: Managing Resistance to Agro-chemicals: From Fundamental Research to Practical Strategies. Green, M. B., Moberg, W. K. and LeBaron, H. (Eds. ). American Chemical Society, Washington, D. C.

    Chapter  Google Scholar 

  6. Sekar, V., Thompson, D.V . Maroney, M.J., Bookland,, R.G. . Adang,M.J. 1987. Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc. Natl. Acad Sci USA 84: 7036–7040.

    Article  CAS  Google Scholar 

  7. De-Souza, M.T., Lecadet, M.-M., Lereclus, D. .1993. Full expression of the crylllA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription. J. Barteriol. 175: 2952–2960.

    Article  CAS  Google Scholar 

  8. Adams, L.F., Brown, K.L., Whiteley, H.R. 1991. Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter. J. Bacteriol 173: 3846–3854.

    Article  CAS  Google Scholar 

  9. Lereclus, D., Delécluse, A., Lecadet, M.-M. 1993. Diversity of Bacillus thuringiensis toxins and genes, 37–69. In: Bacillus thuringiensis an Environmental Biopesticide: Theory and Practice. Entwistle, P. F., Cory, J. S., Bailey, M. J. and Higgs, S. (Eds. ). John Wiley & Sons Ltd, Chichester, UK.

    Google Scholar 

  10. Agaisse, H., Lereclus, D. .1994. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis. Mol. Microbiol. 13: 97–107.

    Article  CAS  Google Scholar 

  11. Agaisse, H., Lereclus, D. 1994. Expression in Bacillus subtilisof the Bacillus thuringiensis crylllA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spoOA mutant. J. Bacteriol. 176: 4734–4741.

    Article  CAS  Google Scholar 

  12. Malvar, T., Baum J. 1994. Tn5401 disruption of the spoOF gene, identified by direct chromosomal sequencing, results in crylllA overproduction in Bacillus thuringiensis.J. Bacteriol. 176: 4750–4753.

    Article  CAS  Google Scholar 

  13. Adams, L.F., Thomas, M.D. 1993. Elucidation of the mechanism of CrylHA hyperexpression in a gamma-irradiated strain of B.thuringiensis subsp. tenebrionis, p. 63. Seventh International Conference on Bacillus. Institut Pasteur, Paris, France.

  14. Malvar, T., Gawron-Burke, C., Baum, J. .1994. Identification of HknA: a KinA-like protein capable of bypassing early Spo mutations that result in CrylllA overproduction in Bacillus thuringiensis. J. Bacteriol. 176: 4742–4749.

    Article  CAS  Google Scholar 

  15. Hoch, J.A. 1993. spo genes, the phosphorelay. and the initiation of sporulation. 747–755. In: Bacillus subtilis and Other Gram-Positive Bacteria. Sonenshein, A. L., Hoch, J. A. and Losick, R. (Eds. ). American Society for Microbiology, Washington, D. C.

    Google Scholar 

  16. Lecadet, M.-M., Chaufaux, J., Lereclus, D. 1992. Construction of novel Bacillus thuringiensis strains with different insecticidal specificities by transduction and by transformation. Appl. Environ. Microbiol. 58: 840–849.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Ferrari, F., Trach, K., Lecoq, D.,, Spence, J., Ferrari, E., Hoch, J. A. 1985. Characterization of the spoOA locus and its deduced product. Proc. Natl. Acad. Sci. USA 82: 2647–2651.

    Article  CAS  Google Scholar 

  18. Arantes, O., Lereclus, D. 1991. Construction of cloning vectors for Bacillus thuringiensis. Gene 108: 115–119.

    Article  CAS  Google Scholar 

  19. Gonzy-Tréboul, G., Karmazyn-Campelli, C., Stragier, P. 1992. Developmental regulation of transcription of the Bacillus subtilis fisAZ operon. J. Mol. Biol. 224: 967–979.

    Article  Google Scholar 

  20. van Hoy, B.E., Hoch, J.A. 1990. Characterization of the spoIVB and recN loci of Bacillus subtilis. J. Bacteriol. 172: 1306–1311.

    Article  CAS  Google Scholar 

  21. Lereclus, D., Vallade, M., Chaufaux, J., Arantes, O., Rambaud, S. . 1992. Expansion of insecticidal host range of Bacillus thuringiensis by in vivo genetic recombination. Bio/Technology 10: 418–421.

    Article  CAS  Google Scholar 

  22. Delécluse, A., Charles, J.-F., Klier, A., Rapoport, G. .1991. Deletion by in vivo recombination shows that the 28-kiloDalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity. J. Bacteriol. 173: 3374–3381.

    Article  Google Scholar 

  23. Brehm, S.P., Staal, S.P., Hoch, J.A. 1973. Phenotypes of pleiotropicnegative sporulation mutants of Bacillus subtilis. J. Bacteriol. 115: 1063–1070

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lereclus, D.,, Arantes, O., Chaufaux, J., Lecadet, M.-M. 1989. Transformation and expression of a cloned δ-endottxin gene in Bacillus thuringiensis FEMS Microbiol. Lett. 60: 211–218.

    Google Scholar 

  25. Chaufeux, J., Marchal M., Müller-Cohn, J., Lereclus, D. 1993. Cloning and expression of a Bacillus thuringiensis gene encoding a δ-entotoxin active against coleopteran larvae. C. R. Acad. Agri. Fr 79: 103–110.

    Google Scholar 

  26. Gibson, T.J. 1984. Studies on the Epstein-Barr virus genome. Ph. D. thesis. University of Cambridge, Cambridge UK.

    Google Scholar 

  27. Lecadet, M.M., Blondel, M.O., Ribier, J. 1980. Generalized transduction in Bacillus thuringiensis var. berliner 1715, using bacteriophage CP54 Ber. J Gen. Microbiol. 121: 203–212.

    Google Scholar 

  28. Villafane, R., Bechhofer, D.H., Narayanan, C.S., Dubnau, D. .1987. Replication control genes of plasmid pE194. J. Bacteriol. 169: 4822–4829.

    Article  CAS  Google Scholar 

  29. Trieu-Cuot, P., Labigne-Roussel, A., Courvalin, P. 1983. An IS15 insertion generates an eight-base-pair duplication of the target DNA. Gene 24: 125–129.

    Article  CAS  Google Scholar 

  30. Msadek, T., Kunst, F., Henner, D., Klier, A., Rapoport, G., Dedonder, R. 1990. Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degV. J. Bacteriol. 172: 824–834.

    Article  CAS  Google Scholar 

  31. Sanger, F., NicHen, S., Coulson, A. R. 1977. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.

    Article  CAS  Google Scholar 

  32. Msadek, T., Kunst, P., Klier, A., Rapoport, G. .1991. DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pieiotropic regulatory gene degQ. J. Bacteriol. 173: 2366–2377.

    Article  CAS  Google Scholar 

  33. Kunst, F., Msadek, T., Rapoport, G. . 1994. Signal Transduction network controlling degradative enzyme synthesis and competence in Bacillus subtilis, 1–19. In: Regulation of Bacterial Differentiation. Piggot, P. (Eds. ). American Society for Microbiology, Washington, D. C.

    Google Scholar 

  34. 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.

    Article  CAS  Google Scholar 

  35. Sanchis, V., Lereclus, D., Menou, G., Chaufaux, J., Lecadet, M.-M. . 1988. Multiplicity of δ-endotoxin genes with different specificities in Bacillus thuringiensis aizawai 7. 29. Mol. Microbiol. 2: 393–404.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Unité de Biochimie Microbienne, URA 1300, Centre National de la Recherche Scientifique, Institut Pasteur, 25 rue du Docteur Roux, 75724, Paris, cedex 15, France

    Didier Lereclus, Hervé Agaisse & Myriam Gominet

  2. Station de Recherche de Lutte Biologique, Institut National de la Recherche Agronomique, La Miniere, 78285, Guyancourt cedex, France

    Didier Lereclus & Josette Chaufaux

Authors
  1. Didier Lereclus
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  2. Hervé Agaisse
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  3. Myriam Gominet
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  4. Josette Chaufaux
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Correspondence to Didier Lereclus.

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Lereclus, D., Agaisse, H., Gominet, M. et al. Overproduction of Encapsulated Insecticidal Crystal Proteins in a Bacillus thuringiensis spoOA Mutant. Nat Biotechnol 13, 67–71 (1995). https://doi.org/10.1038/nbt0195-67

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