Figure 1
Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ sp. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p/STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1. (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc)3 for detection. Lane 1, E. coli- pEHchiA74∆sp; lane 2, without sample; lane 3, E. coli- pEBchiA74∆sp; lane 4, E. coli DH5α. Black arrow indicates the position of ChiA74∆sp in recombinant E. coli strains. Panel 2: (a) E. coli- pEHchiA74∆sp, (b) E. coli- pEBchiA74∆sp. No endochitinase activity was observed with E. coli DH5α. (C) Phase contrast microscopy of recombinant strains of B. thuringiensis. Panel 1, 4Q7; panel 2, 4Q7- pEHchiA74∆sp; panel 3, 4Q7- pEBchiA74∆sp. Black arrows indicate the positions of ChiA74∆sp inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7- pEHchiA74∆sp; lane 3, 4Q7- pEBchiA74∆sp. Black arrow indicates the position of ChiA74∆sp in recombinant 4Q7 strains. Panel 2: (a) 4Q7- pEHchiA74∆sp, (b) 4Q7- pEBchiA74∆sp. Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.