Abstract
The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.
Full Text
The Full Text of this article is available as a PDF (233.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adang M. J., Staver M. J., Rocheleau T. A., Leighton J., Barker R. F., Thompson D. V. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene. 1985;36(3):289–300. doi: 10.1016/0378-1119(85)90184-2. [DOI] [PubMed] [Google Scholar]
- Bradley D., Harkey M. A., Kim M. K., Biever K. D., Bauer L. S. The insecticidal CryIB crystal protein of Bacillus thuringiensis ssp. thuringiensis has dual specificity to coleopteran and lepidopteran larvae. J Invertebr Pathol. 1995 Mar;65(2):162–173. doi: 10.1006/jipa.1995.1024. [DOI] [PubMed] [Google Scholar]
- Büttcher V., Rühlmann A., Cramer F. Improved single-stranded DNA producing expression vectors for protein manipulation in Escherichia coli. Nucleic Acids Res. 1990 Feb 25;18(4):1075–1075. doi: 10.1093/nar/18.4.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crickmore N., Ellar D. J. Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis. Mol Microbiol. 1992 Jun;6(11):1533–1537. doi: 10.1111/j.1365-2958.1992.tb00874.x. [DOI] [PubMed] [Google Scholar]
- Donovan W. P., Dankocsik C. C., Gilbert M. P., Gawron-Burke M. C., Groat R. G., Carlton B. C. Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki. J Biol Chem. 1988 Jan 5;263(1):561–567. [PubMed] [Google Scholar]
- Haider M. Z., Ellar D. J. Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture. Mol Microbiol. 1987 Jul;1(1):59–66. doi: 10.1111/j.1365-2958.1987.tb00527.x. [DOI] [PubMed] [Google Scholar]
- Haider M. Z., Ellar D. J. Nucleotide sequence of a Bacillus thuringiensis aizawai IC1 entomocidal crystal protein gene. Nucleic Acids Res. 1988 Nov 25;16(22):10927–10927. doi: 10.1093/nar/16.22.10927. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haider M. Z., Knowles B. H., Ellar D. J. Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases. Eur J Biochem. 1986 May 2;156(3):531–540. doi: 10.1111/j.1432-1033.1986.tb09612.x. [DOI] [PubMed] [Google Scholar]
- Hodgman T. C., Ellar D. J. Models for the structure and function of the Bacillus thuringiensis delta-endotoxins determined by compilational analysis. DNA Seq. 1990;1(2):97–106. doi: 10.3109/10425179009016037. [DOI] [PubMed] [Google Scholar]
- Honée G., van der Salm T., Visser B. Nucleotide sequence of crystal protein gene isolated from B. thuringiensis subspecies entomocidus 60.5 coding for a toxin highly active against Spodoptera species. Nucleic Acids Res. 1988 Jul 11;16(13):6240–6240. doi: 10.1093/nar/16.13.6240. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., van Montagu M., Zabeau M. Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem. 1986 Dec 1;161(2):273–280. doi: 10.1111/j.1432-1033.1986.tb10443.x. [DOI] [PubMed] [Google Scholar]
- Knight P. J., Crickmore N., Ellar D. J. The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol Microbiol. 1994 Feb;11(3):429–436. doi: 10.1111/j.1365-2958.1994.tb00324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Knowles B. H., Ellar D. J. Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin. J Cell Sci. 1986 Jul;83:89–101. doi: 10.1242/jcs.83.1.89. [DOI] [PubMed] [Google Scholar]
- Knowles B. H., Ellar D. J. Differential specificity of two insecticidal toxins from Bacillus thuringiensis var. aizawai. Mol Microbiol. 1988 Jan;2(1):153–157. doi: 10.1111/j.1365-2958.1988.tb00016.x. [DOI] [PubMed] [Google Scholar]
- Knowles B. H., Francis P. H., Ellar D. J. Structurally related Bacillus thuringiensis delta-endotoxins display major differences in insecticidal activity in vivo and in vitro. J Cell Sci. 1986 Aug;84:221–236. doi: 10.1242/jcs.84.1.221. [DOI] [PubMed] [Google Scholar]
- Knowles B. H., Thomas W. E., Ellar D. J. Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action. FEBS Lett. 1984 Mar 26;168(2):197–202. doi: 10.1016/0014-5793(84)80245-8. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lieberman H. R. Estimating LD50 using the probit technique: a BASIC computer program. Drug Chem Toxicol. 1983;6(1):111–116. doi: 10.3109/01480548309072465. [DOI] [PubMed] [Google Scholar]
- Sanchis V., Lereclus D., Menou G., Chaufaux J., Guo S., Lecadet M. M. Nucleotide sequence and analysis of the N-terminal coding region of the Spodoptera-active delta-endotoxin gene of Bacillus thuringiensis aizawai 7.29. Mol Microbiol. 1989 Feb;3(2):229–238. doi: 10.1111/j.1365-2958.1989.tb01812.x. [DOI] [PubMed] [Google Scholar]
- Sanchis V., Lereclus D., Menou G., Chaufaux J., Lecadet M. M. Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29. Mol Microbiol. 1988 May;2(3):393–404. doi: 10.1111/j.1365-2958.1988.tb00044.x. [DOI] [PubMed] [Google Scholar]
- Schnepf H. E., Wong H. C., Whiteley H. R. The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol Chem. 1985 May 25;260(10):6264–6272. [PubMed] [Google Scholar]
- Smith G. P., Ellar D. J., Keeler S. J., Seip C. E. Nucleotide sequence and analysis of an insertion sequence from Bacillus thuringiensis related to IS150. Plasmid. 1994 Jul;32(1):10–18. doi: 10.1006/plas.1994.1039. [DOI] [PubMed] [Google Scholar]
- Smith G. P., Ellar D. J. Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC delta-endotoxin affects insecticidal specificity. Biochem J. 1994 Sep 1;302(Pt 2):611–616. doi: 10.1042/bj3020611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stewart G. S., Johnstone K., Hagelberg E., Ellar D. J. Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem J. 1981 Jul 15;198(1):101–106. doi: 10.1042/bj1980101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tailor R., Tippett J., Gibb G., Pells S., Pike D., Jordan L., Ely S. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidal to coleopteran and lepidopteran larvae. Mol Microbiol. 1992 May;6(9):1211–1217. doi: 10.1111/j.1365-2958.1992.tb01560.x. [DOI] [PubMed] [Google Scholar]
- Thomas W. E., Ellar D. J. Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J Cell Sci. 1983 Mar;60:181–197. doi: 10.1242/jcs.60.1.181. [DOI] [PubMed] [Google Scholar]
- Widner W. R., Whiteley H. R. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J Bacteriol. 1989 Feb;171(2):965–974. doi: 10.1128/jb.171.2.965-974.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
