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Applied and Environmental Microbiology, June 2007, p. 3623-3629, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.01056-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation{triangledown}

Jesko Oestergaard,1 Ralf-Udo Ehlers,1* Amparo C. Martínez-Ramírez,2 and Maria Dolores Real2

Institute for Phytopathology, Department of Biotechnology and Biological Control, Christian Albrechts University, Kiel, Germany,1 Departamento de Genetica, Facultat de Ciències Biològiques, Universitat de Valencia, Burjassot, Valencia, Spain2

Received 8 May 2006/ Accepted 27 March 2007

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.

* Corresponding author. Mailing address: Institute for Phytopathology, Department of Biotechnology and Biological Control, Christian Albrechts University, Hermann-Rodewald Str. 9, 24118 Kiel, Germany. Phone: 49 (0)431 880 4864. Fax: 49 (0)431 880 1583. E-mail: ehlers{at}biotec.uni-kiel.de

{triangledown} Published ahead of print on 6 April 2007.

Applied and Environmental Microbiology, June 2007, p. 3623-3629, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.01056-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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