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 Manoj Kumar, A. S.
Right arrow Articles by Aronson, A. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Manoj Kumar, A. S.
Right arrow Articles by Aronson, A. I.

 Previous Article  |  Next Article 

Journal of Bacteriology, October 1999, p. 6103-6107, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Analysis of Mutations in the Pore-Forming Region Essential for Insecticidal Activity of a Bacillus thuringiensis delta -Endotoxin

A. S. Manoj Kumar and A. I. Aronson*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received 8 March 1999/Accepted 29 July 1999

The Bacillus thuringiensis insecticidal delta -endotoxins have a three-domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the B. thuringiensis cry1Ac1 gene, encoding parts of helix alpha 4 and the loop connecting helices alpha 4 and alpha 5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site-directed mutations were also created in specific surface residues of helix alpha 3 in order to increase its hydrophobicity. Among 12 random mutations in helix alpha 4, 5 resulted in the total loss of toxicity for Manduca sexta and Heliothis virescens, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices alpha 4 and alpha 5 did not affect toxicity, nor did mutations in alpha 3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix alpha 5, those in helix alpha 4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix alpha 5 is important for oligomerization and perhaps has other functions, whereas helix alpha 4 must have a more direct role in establishing the properties of the channel.

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, W. Lafayette, IN 47906. Phone: (765) 494 4992. Fax: (765) 494 0876. E-mail: aaronson{at}bilbo.bio.purdue.edu.

Journal of Bacteriology, October 1999, p. 6103-6107, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Lebel, G., Vachon, V., Prefontaine, G., Girard, F., Masson, L., Juteau, M., Bah, A., Larouche, G., Vincent, C., Laprade, R., Schwartz, J.-L. (2009). Mutations in Domain I Interhelical Loops Affect the Rate of Pore Formation by the Bacillus thuringiensis Cry1Aa Toxin in Insect Midgut Brush Border Membrane Vesicles. Appl. Environ. Microbiol. 75: 3842-3850 [Abstract] [Full Text]  
  • Girard, F., Vachon, V., Prefontaine, G., Marceau, L., Schwartz, J.-L., Masson, L., Laprade, R. (2009). Helix {alpha}4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation. Appl. Environ. Microbiol. 75: 359-365 [Abstract] [Full Text]  
  • Girard, F., Vachon, V., Prefontaine, G., Marceau, L., Su, Y., Larouche, G., Vincent, C., Schwartz, J.-L., Masson, L., Laprade, R. (2008). Cysteine Scanning Mutagenesis of {alpha}4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa. Appl. Environ. Microbiol. 74: 2565-2572 [Abstract] [Full Text]  
  • Zhang, X., Candas, M., Griko, N. B., Taussig, R., Bulla, L. A. Jr. (2006). A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proc. Natl. Acad. Sci. USA 103: 9897-9902 [Abstract] [Full Text]  
  • Vachon, V., Prefontaine, G., Rang, C., Coux, F., Juteau, M., Schwartz, J.-L., Brousseau, R., Frutos, R., Laprade, R., Masson, L. (2004). Helix 4 Mutants of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities. Appl. Environ. Microbiol. 70: 6123-6130 [Abstract] [Full Text]  
  • Cuconati, A., White, E. (2002). Viral homologs of BCL-2: role of apoptosis in the regulation of virus infection. Genes Dev. 16: 2465-2478 [Full Text]  
  • Tigue, N. J., Jacoby, J., Ellar, D. J. (2001). The alpha -Helix 4 Residue, Asn135, Is Involved in the Oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis Toxins. Appl. Environ. Microbiol. 67: 5715-5720 [Abstract] [Full Text]  
  • Sundararajan, R., White, E. (2001). E1B 19K Blocks Bax Oligomerization and Tumor Necrosis Factor Alpha-Mediated Apoptosis. J. Virol. 75: 7506-7516 [Abstract] [Full Text]  
  • Aronson, A. (2000). Incorporation of Protease K into Larval Insect Membrane Vesicles Does Not Result in Disruption of Integrity or Function of the Pore-Forming Bacillus thuringiensis delta -Endotoxin. Appl. Environ. Microbiol. 66: 4568-4570 [Abstract] [Full Text]  
  • Gerber, D., Shai, Y. (2000). Insertion and Organization within Membranes of the delta -Endotoxin Pore-forming Domain, Helix 4-Loop-Helix 5, and Inhibition of Its Activity by a Mutant Helix 4 Peptide. J. Biol. Chem. 275: 23602-23607 [Abstract] [Full Text]  
  • Sundararajan, R., Cuconati, A., Nelson, D., White, E. (2001). Tumor Necrosis Factor-alpha Induces Bax-Bak Interaction and Apoptosis, Which Is Inhibited by Adenovirus E1B 19K. J. Biol. Chem. 276: 45120-45127 [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»