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

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2000, p. 5174-5181, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Role of Proteolysis in Determining Potency of Bacillus thuringiensis Cry1Ac delta -Endotoxin

Daniel J. Lightwood,1,dagger David J. Ellar,1,* and Paul Jarrett2

Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA,1 and Horticulture Research International, Wellesbourne, Warwickshire, CV35 9EF,2 United Kingdom

Received 10 April 2000/Accepted 18 September 2000

Bacillus thuringiensis protein delta -endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta -endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of ~56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of ~58, ~40, and ~20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.

* Corresponding author. Mailing address: Department of Biochemistry, Old Addenbrookes Site, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom. Phone: 44 (0) 1223 333651. Fax: 44 (0) 1223 766043. E-mail: djel{at}mole.bio.cam.ac.uk.

dagger Present address: Celltech Chiroscience plc, Slough, Berkshire, SL1 4EN, United Kingdom.

Applied and Environmental Microbiology, December 2000, p. 5174-5181, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Anilkumar, K. J., Rodrigo-Simon, A., Ferre, J., Pusztai-Carey, M., Sivasupramaniam, S., Moar, W. J. (2008). Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie). Appl. Environ. Microbiol. 74: 462-469 [Abstract] [Full Text]  
  • Walters, F. S., Stacy, C. M., Lee, M. K., Palekar, N., Chen, J. S. (2008). An Engineered Chymotrypsin/Cathepsin G Site in Domain I Renders Bacillus thuringiensis Cry3A Active against Western Corn Rootworm Larvae. Appl. Environ. Microbiol. 74: 367-374 [Abstract] [Full Text]  
  • Fortier, M., Vachon, V., Frutos, R., Schwartz, J.-L., Laprade, R. (2007). Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins. Appl. Environ. Microbiol. 73: 6208-6213 [Abstract] [Full Text]  
  • Kirouac, M., Vachon, V., Quievy, D., Schwartz, J.-L., Laprade, R. (2006). Protease Inhibitors Fail To Prevent Pore Formation by the Activated Bacillus thuringiensis Toxin Cry1Aa in Insect Brush Border Membrane Vesicles. Appl. Environ. Microbiol. 72: 506-515 [Abstract] [Full Text]  
  • Sayyed, A. H., Gatsi, R., Ibiza-Palacios, M. S., Escriche, B., Wright, D. J., Crickmore, N. (2005). Common, but Complex, Mode of Resistance of Plutella xylostella to Bacillus thuringiensis Toxins Cry1Ab and Cry1Ac. Appl. Environ. Microbiol. 71: 6863-6869 [Abstract] [Full Text]  
  • Sayyed, A. H., Gatsi, R., Kouskoura, T., Wright, D. J., Crickmore, N. (2001). Susceptibility of a Field-Derived, Bacillus thuringiensis-Resistant Strain of Diamondback Moth to In Vitro-Activated Cry1Ac Toxin. Appl. Environ. Microbiol. 67: 4372-4373 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»