Abstract
Previous studies have shown that a 20-kDa protein enhances production of the insecticidal CytA and CryIVA proteins of Bacillus thuringiensis in Escherichia coli as well as CytA production and crystal formation in B. thuringiensis. To determine whether the 20-kDa protein could enhance CryIVD production, an expression vector was constructed with the 20-kDa open-reading frame under control of cryIA(c) promoters and the cryIVD gene under control of its own promoter. Acrystalliferous cells of B. thuringiensis transformed with this plasmid, designated pWF53, produced large bitrapezoidal CryIVD crystals that averaged 1.3 × 0.92 × 0.31 μm, approximately fivefold larger in volume than wild-type CryIVD crystals, and 1.7 fold the volume of crystals produced using the cryIV D operon, which contains the cryIV D gene and the gene for the 20-kDa protein. These results demonstrate that the 20-kDa protein significantly improves net synthesis of CryIVD and promotes CryIVD crystal formation. Improved production of proteins as diverse as CryIVD and CytA by the 20-kDa protein indicates this protein may be useful in facilitating the production of other proteins.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adams LF, Visick JE, Whiteley HR (1989) A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli. J Bacteriol 171:521–530
Arvidson H, Dunn PE, Strnad S, Aronson AI (1989) Specificity of Bacillus thuringiensis for lepidopteran larvae: factors involved in vivo and in the structure of a purified protoxon. Mol Microbiol 3:1533–1543
Crickmore N, Bone EJ, Ellar DJ (1990) Genetic manipulation of Bacillus thuringiensis: towards an improved pesticide. Aspects Appl Biol 24:17–24
Donovan WP, Dankocsik C, Gilbert P (1988) Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis. J Bacteriol 170:4732–4738
Galjart NJ, Sivasubramanian N, Federici BA (1987) Plasmid location, cloning, and sequence analysis of the gene encoding a 27.3-kDa cytolytic protein from Bacillus thuringiensis subsp. morrisoni (PG-14). Curr Microbiol 16:171–177
Goldberg LJ, Margalit J (1977) A bacterial spore demonstrating rapid larvicidal activity against Anopheles segentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti, and Culex pipiens. Mosq News 37:355–358
Hofte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255
Ibarra JE, Federici BA (1986a) Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity. FEMS Microbiol Lett 34:79–84
Ibarra JE, Federici BA (1986b) Isolation of a relatively non-toxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J Bacteriol 165:527–533
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Lereclus D, Arantes O, Chaufaux J, Lecadet MM (1989) Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 60:211–218
Padua LE, Ohba M, Aizawa K (1984) Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae. J Invertebr Pathol 44:12–17
Schuster W, Geiser M, Mathe D (1989) Efficient transformation of Bacillus thuringiensis and B. cereus via electroporation: transformation of crystalliferous strains with a cloned delta-endotoxin gene. Mol Gen Gen 218:177–181
Tyrell DJ, Bulla LA Jr, Andrews RE Jr, Kramer KJ, Davidson LI, Nordin P (1981) Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains. J Bacteriol 145:1052–1062
Visick JE, Whiteley HR (1991) Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli. J Bacteriol 173:1748–1756
Ward ES, Ridley AR, Ellar DJ, Todd JA (1986) Bacillus thuringiensis var israelensis δ-endotoxin: cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J Mol Biol 191:13–22
Wu D, Aronson AI (1992) Localized mutagenesis defines regions of the Bacillus thuringiensis δ-endotoxin involved in toxicity and specificity. J Biol Chem 267:2311–2317
Wu D, Federici BA (1993) A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J Bacteriol 175:5276–5280
Wu D, Cao XL, Bai YY, Aronson AI (1991) Sequence of an operon containing a novel delta-endotoxin gene from Bacillus thuringiensis. FEMS Microbiol Lett 81:31–36
Yoshisue H, Yoshida K, Sen K, Sakai H, Komano T (1992) Effects of Bacillus thuringiensis var. israelensis 20-kDa protein on production of the Bti 130-kDa crystal protein in Escherichia coli. Biophys Biotechnol Biochem 56:1429–1433
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wu, D., Federici, B.A. Improved production of the insecticidal CryIVD protein in Bacillus thuringiensis using cryIA(c) promoters to express the gene for an associated 20-kDa protein. Appl Microbiol Biotechnol 42, 697–702 (1995). https://doi.org/10.1007/BF00171947
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00171947