Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities

• Published in: Applied and Environmental Microbiology 2004-10, Vol.70 (10), p.6123-6130
• Main Authors: Vachon, V, Prefontaine, G, Rang, C, Coux, F, Juteau, M, Schwartz, J.L, Brousseau, R, Frutos, R, Laprade, R, Masson, L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Bacillus thuringiensis; Bacillus thuringiensis - genetics; Bacillus thuringiensis - physiology; Bacteria; bacterial insecticides; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - physiology; Bacterial Proteins - toxicity; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Bacterial Toxins - toxicity; brush border membrane vesicles; Cell Membrane - drug effects; cell membranes; crystal proteins; cytotoxicity; Endotoxins - chemistry; Endotoxins - genetics; Endotoxins - physiology; Endotoxins - toxicity; Hemolysin Proteins; In Vitro Techniques; insect pests; insecticidal properties; insecticidal proteins; Insecticides; Invertebrate Microbiology; Kinetics; Manduca - drug effects; Manduca sexta; membrane permeability; Microvilli - drug effects; Molecular Sequence Data; Mutagenesis, Site-Directed; mutants; Mutation; Pest Control, Biological; Protein Structure, Secondary; site-directed mutagenesis; Toxins
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.70.10.6123-6130.2004

The role played by alpha-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that alpha-helix 4 plays a crucial role in the mechanism of pore formation.