Mapping and characterization of the entomocidal domain of the Bacillus thuringiensis CryIA(b) protoxin.
The amino acid sequences necessary for entomocidal activity of the CryIA(b) protoxin of Bacillus thuringiensis were determined. Introduction of stop codons behind codons Arg601, Phe604 or Ala607 showed that amino acid residues C-terminal to Ala607 are not required for insecticidal activity and that activation by midgut proteases takes place distal to Ala607. The two shortest polypeptides, deleted for part of the highly conserved beta-strand, were prone to proteolytic degradation, explaining their lack of toxicity. Apparently, this beta-strand is essential for folding of the molecule into a stable conformation. Proteolytic activation at the N-terminus was investigated by removing the first 28 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 acid residues 29-429, encompassing the complete putative pore forming domain, but lacking a large part of the receptor-binding domain, was similarly toxic to E. coli cells. This suggests a role for the N-terminal 28 amino acids in rendering the toxin inactive in Bacillus thuringiensis, and indicates that the cytolytic potential of the pore forming domain 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 cleaved by trypsin in vitro, and reduced its toxicity to M. sexta larvae.
Martens
J W
JW
Department of Virology, Agricultural University Wageningen, The Netherlands.
Visser
B
B
Vlak
J M
JM
Bosch
D
D
D000818:Animals; D001413:Bacillus thuringiensis / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins; D001483:Base Sequence; D001681:Biological Assay; D017931:DNA Primers / Q000737:chemistry; D004731:Endotoxins / Q000737:chemistry*; D006460:Hemolysin Proteins; D018402:Manduca / Q000187:drug effects; D008969:Molecular Sequence Data; D009036:Moths / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis