MAPPING AND CHARACTERIZATION OF THE ENTOMOCIDAL DOMAIN OF THE BACILLUS-THURINGIENSIS CRYIA(B) PROTOXIN

The amino acid sequences necessary for entomocidal activity of the Cry IA(b) protoxin of Bacillus thuringiensis were determined. Introduction of stop codons behind codons Arg601, Phe604 or Ala607 showed that ami no acid residues C-terminal to Ala607 are not required for insecticida l activity and that activation by mid,aut proteases takes place distal to Ala607. The two shortest polypeptides, deleted for part of the hig hly conserved beta-strand, were prone to proteolytic degradation, expl aining their lack of toxicity. Apparently, this beta-strand is essenti al for folding of the molecule into a stable conformation. Proteolytic activation at the N-terminus was investigated by removing the first 2 8 codons, resulting in a translation product extending from amino acid 29 to 607. This protein appeared to be toxic not only to susceptible insect larvae such as Manduca sexta and Heliothis virescens, but also to Escherichia coli cells. An additional mutant, encoding only amino a cid residues 29-429, encompassing the complete putative pore forming d omain, but lacking a large part of the receptor-binding domain, was si milarly toxic to E. coli cells. This suggests a role for the N-termina l 28 amino acids in rendering the toxin inactive in Bacillus thuringie nsis, and indicates that the cytolytic potential of the pore forming d omain is only realized after proteolytic removal of these residues by proteases in the insect gut. In line with this hypothesis are results obtained with a mutant protein in which Arg28 at the cleavage site was replaced by Asp. This substitution prevented the protein from being c leaved by trypsin in vitro, and reduced its toxicity to M. sexta larva e.