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Applied and Environmental Microbiology, December 2008, p. 7145-7151, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.00752-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins{triangledown}

Samir Naimov,1* Rumyana Boncheva,1 Rumyana Karlova,2 Stefan Dukiandjiev,1 Ivan Minkov,1 and Ruud A. de Maagd3

Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24 Tzar Assen Street, 4000 Plovdiv, Bulgaria,1 Department of Biochemistry, Wageningen University and Research Centre, P.O. Box 8128, 6700 ET Wageningen, The Netherlands,2 Business Unit Bioscience, Plant Research International B.V., Wageningen University and Research Centre, P.O. Box 16, 6700 AA Wageningen, The Netherlands3

Received 31 March 2008/ Accepted 23 September 2008

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

* Corresponding author. Mailing address: University of Plovdiv, 24 Tzar Assen Street, 4000 Plovdiv, Bulgaria. Phone and fax: 35932620944. E-mail: naimov0{at}uni-plovdiv.bg

{triangledown} Published ahead of print on 3 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7145-7151, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.00752-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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