Figure 4
Expression of ChiA74Δsp in B. thuringiensis HD1. (A) Phase contrast micrograph of HD1 sporangia, (B) HD1-pEBchiA74∆sp sporangia, (C) HD1-pEBchiA74∆sp lysed culture. Triangle and triangle-wide wavy arrows indicate the presence of Cry crystals and ChiA74∆sp inclusion bodies, respectively. In (A) arrow shows a bipyramidal crystal (Cry1) associated with a cuboidal crystal (Cry2). ChiA74∆sp was fused to the GFP and expressed in HD1 to obtain HD1-pEBchiA74∆sp-gfp; (D) Phase contrast, and (E) fluorescence microscopy, black and white arrows indicate the crystal position. Fluorescence along the sporangium indicates that ChiA74∆sp was expressed and accumulated within the cell. (F) Confirmation of the transformation of HD1 by PCR. Lanes 1 and 5, HD1; Lanes 2 and 6, HD1-pEBchiA74∆sp; Lanes 3 and 7, E. coli-pEBchiA74Δsp; lanes 4 and 8, pBluescript KS (+) (Stratagene); L, 1 kb DNA Ladder (10, 8, 6, 5, 4, 3, 2, 1.5, 1 kb; New England Biolabs). Amplicons of lanes 1 to 4 and 5 to 8 were obtained using the primers ery1, ery2, and cytSTAB-1, chiA74-4, respectively. (G) SDS-PAGE and (H) zymogram of solubilized proteins obtained from sporulated and lysed HD1 (lane 1) and HD1-pEBchiA74∆sp (lane 2). Location of Cry1A, Cry2A proteins and endochitinase (ChiA74∆sp) are showed with black arrows. A reduction in the relative amount of Cry1 (~133 kDa) and Cry2A (~65 kDa) proteins was observed in the recombinant strain (lane 2) compared with wildtype HD1 (lane1). Zymogram detection was performed using the 4-MU-(GlcNAc)3 substrate. Asterisks indicate probable ChiA74∆sp degradation products. Protein molecular masses were deduced using the reference BenchMark protein ladder standard (Invitrogen, Carlsbad CA, USA).