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Expression of toxin genes from Bacillus thuringiensis subsp. Thompsoni in operon fusion with Orf-1 and Orf-2 leads to the formation of parasporal bodies

Rang C., Bes M., William J., Moar W.J., Frutos R.. 1994. In : SIP. 6th international colloquium on invertebrate pathology and microbial control incorporating the 2nd international conference on Bacillus thuringiensis. Abstracts. Bethesda : SIP, p. 31-31. International colloquium on invertebrate pathology and microbial control. 6, 1994-08-28/1994-09-02, Montpellier (France).

Orf-1 and orf-2 are associated in operon with the cryIIA gene and their products appear to be essential for the crystallization of CryIIA most likely by acting as molecular chaperones. A novel strain of Bacillus thuriagiensis subsp. thompsoni named HnC produces during sporulation pyramidal crystals that are composed of two proteins of 38 kDa and 40 kDa. The two genes coding for the 38 kDa and 40 kDa proteins were isolated from HnC and placed under the control of the cryIIA promoter from NRD-12. Similary, these genes were cloned downstream from the cryIIA promoter, orf-1 and orf-2 in place of cryIIA. These constructs were transfered into a crystal-minus strain of B. thuringiensis subsp. Kurstaki HD-1 using the shuttle vector pHT 3101. Expression of the 38-kDa or the 40-kDa protein gene under the control of the CryIIA promoter, in HD-1 cry(-) cells resulted in low level of production of protein without formation of inclusion bodies. Bacteria expressing the two genes show the same characteristics. In contrary, simultaneous expression of each gene with orf-1 and orf-2 in HD-1cry(-) cells allowed accumulation of both 38 kDa or 40 kDa proteins in parasporal inclusion bodies visible under light microscopy. Analysis of these inclusions by SDS-PAGE and immunodetection demonstrated the presence of the 38 kDa and 40 kDa proteins which migrated in SDS-PAGE to the same position than crystal proteins from HnC. These results demonstrate that orf-1 and orf-2 can direct the crystallization of toxins with no homology to CryII proteins.

Mots-clés : bacillus thuringiensis; expression des gènes; toxine; génie génétique

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