This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Maagd, R. A. Articles by Bosch, D. Search for Related Content PubMed PubMed Citation Articles by de Maagd, R. A. Articles by Bosch, D. Agricola Articles by de Maagd, R. A. Articles by Bosch, D.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., 05 1996, 1537-1543, Vol 62, No. 5
Copyright © 1996, American Society for Microbiology

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition

RA de Maagd, MS Kwa, H van der Klei, T Yamamoto, B Schipper, JM Vlak, WJ Stiekema and D Bosch
Department of Molecular Biology, Centre for Plant Breeding and Reproduction Research, Wageningen, The Netherlands. R.A.deMaagd@CPRO.DLO.NL

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin- labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.

This article has been cited by other articles:

• Pigott, C. R., King, M. S., Ellar, D. J. (2008). Investigating the Properties of Bacillus thuringiensis Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology. Appl. Environ. Microbiol. 74: 3497-3511 [Abstract] [Full Text]  
• Fang, J., Xu, X., Wang, P., Zhao, J.-Z., Shelton, A. M., Cheng, J., Feng, M.-G., Shen, Z. (2007). Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins. Appl. Environ. Microbiol. 73: 956-961 [Abstract] [Full Text]  
• Boonserm, P., Mo, M., Angsuthanasombat, C., Lescar, J. (2006). Structure of the Functional Form of the Mosquito Larvicidal Cry4Aa Toxin from Bacillus thuringiensis at a 2.8-Angstrom Resolution. J. Bacteriol. 188: 3391-3401 [Abstract] [Full Text]  
• Liu, X. S., Dean, D. H. (2006). Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin. Protein Eng Des Sel 19: 107-111 [Abstract] [Full Text]  
• Rodrigo-Simon, A., de Maagd, R. A., Avilla, C., Bakker, P. L., Molthoff, J., Gonzalez-Zamora, J. E., Ferre, J. (2006). Lack of Detrimental Effects of Bacillus thuringiensis Cry Toxins on the Insect Predator Chrysoperla carnea: a Toxicological, Histopathological, and Biochemical Analysis. Appl. Environ. Microbiol. 72: 1595-1603 [Abstract] [Full Text]  
• Atsumi, S., Mizuno, E., Hara, H., Nakanishi, K., Kitami, M., Miura, N., Tabunoki, H., Watanabe, A., Sato, R. (2005). Location of the Bombyx mori Aminopeptidase N Type 1 Binding Site on Bacillus thuringiensis Cry1Aa Toxin. Appl. Environ. Microbiol. 71: 3966-3977 [Abstract] [Full Text]  
• Zhuang, M., Oltean, D. I., Gomez, I., Pullikuth, A. K., Soberon, M., Bravo, A., Gill, S. S. (2002). Heliothis virescens and Manduca sexta Lipid Rafts Are Involved in Cry1A Toxin Binding to the Midgut Epithelium and Subsequent Pore Formation. J. Biol. Chem. 277: 13863-13872 [Abstract] [Full Text]  
• Naimov, S., Weemen-Hendriks, M., Dukiandjiev, S., de Maagd, R. A. (2001). Bacillus thuringiensis Delta-Endotoxin Cry1 Hybrid Proteins with Increased Activity against the Colorado Potato Beetle. Appl. Environ. Microbiol. 67: 5328-5330 [Abstract] [Full Text]  
• Hua, G., Masson, L., Jurat-Fuentes, J. L., Schwab, G., Adang, M. J. (2001). Binding Analyses of Bacillus thuringiensis Cry {delta}-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis. Appl. Environ. Microbiol. 67: 872-879 [Abstract] [Full Text]  
• Jurat-Fuentes, J. L., Adang, M. J. (2001). Importance of Cry1 {delta}-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.). Appl. Environ. Microbiol. 67: 323-329 [Abstract] [Full Text]  
• Jenkins, J. L., Lee, M. K., Valaitis, A. P., Curtiss, A., Dean, D. H. (2000). Bivalent Sequential Binding Model of a Bacillus thuringiensis Toxin to Gypsy Moth Aminopeptidase N Receptor. J. Biol. Chem. 275: 14423-14431 [Abstract] [Full Text]  
• de Maagd, R. A., Weemen-Hendriks, M., Stiekema, W., Bosch, D. (2000). Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Can Function as a Specificity Determinant for Spodoptera exigua in Different, but Not All, Cry1-Cry1C Hybrids. Appl. Environ. Microbiol. 66: 1559-1563 [Abstract] [Full Text]  
• Oltean, D. I., Pullikuth, A. K., Lee, H.-K., Gill, S. S. (1999). Partial Purification and Characterization of Bacillus thuringiensis Cry1A Toxin Receptor A from Heliothis virescens and Cloning of the Corresponding cDNA. Appl. Environ. Microbiol. 65: 4760-4766 [Abstract] [Full Text]  
• de Maagd, R. A., Bakker, P., Staykov, N., Dukiandjiev, S., Stiekema, W., Bosch, D. (1999). Identification of Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity. Appl. Environ. Microbiol. 65: 4369-4374 [Abstract] [Full Text]  
• Lee, M. K., You, T. H., Gould, F. L., Dean, D. H. (1999). Identification of Residues in Domain III of Bacillus thuringiensis Cry1Ac Toxin That Affect Binding and Toxicity. Appl. Environ. Microbiol. 65: 4513-4520 [Abstract] [Full Text]  
• Rang, C., Vachon, V., de Maagd, R. A., Villalon, M., Schwartz, J.-L., Bosch, D., Frutos, R., Laprade, R. (1999). Interaction between Functional Domains of Bacillus thuringiensis Insecticidal Crystal Proteins. Appl. Environ. Microbiol. 65: 2918-2925 [Abstract] [Full Text]  
• Ballester, V., Granero, F., de Maagd, R. A., Bosch, D., Ménsua, J. L., Ferré, J. (1999). Role of Bacillus thuringiensis Toxin Domains in Toxicity and Receptor Binding in the Diamondback Moth. Appl. Environ. Microbiol. 65: 1900-1903 [Abstract] [Full Text]  
• Luo, K., Banks, D., Adang, M. J. (1999). Toxicity, Binding, and Permeability Analyses of Four Bacillus thuringiensis Cry1 delta -Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda. Appl. Environ. Microbiol. 65: 457-464 [Abstract] [Full Text]  
• Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R., Dean, D. H. (1998). Bacillus thuringiensis and Its Pesticidal Crystal Proteins. Microbiol. Mol. Biol. Rev. 62: 775-806 [Abstract] [Full Text]  
• Rajamohan, F., Alzate, O., Cotrill, J. A., Curtiss, A., Dean, D. H. (1996). Protein engineering of Bacillus thuringiensis delta -endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae. Proc. Natl. Acad. Sci. USA 93: 14338-14343 [Abstract] [Full Text]  
• Coux, F., Vachon, V., Rang, C., Moozar, K., Masson, L., Royer, M., Bes, M., Rivest, S., Brousseau, R., Schwartz, J.-L., Laprade, R., Frutos, R. (2001). Role of Interdomain Salt Bridges in the Pore-forming Ability of the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac. J. Biol. Chem. 276: 35546-35551 [Abstract] [Full Text]