Identification of amino acids required for receptor binding and toxicity of the Bacillus sphaericus binary toxin

Bacillus sphaericus produces a mosquito-larvicidal binary toxin composed of BinB and BinA subunits. BinA is important for toxicity, whereas BinB acts as a specific receptor-binding component. To study the functional significance of two regions that are only present in BinB, four block mutations and two single mutations were initially introduced: ₁₁₁YLD₁₁₃→ ₁₁₁AAA₁₁₃, ₁₁₅NNH₁₁₇→ ₁₁₅AAA₁₁₇, ₁₄₃GEQ₁₄₅ →₁₄₃AAA₁₄₅, ₁₄₇FQFY₁₅₀→ ₁₄₇AAAA₁₅₀, N114A and F146A. Only the replacements at ₁₄₇FQFY₁₅₀ resulted in a total loss of toxicity to Culex quinquefasciatus larvae. Further single alanine substitutions in this region, F147A, Q148A, F149A and Y150A, were introduced to identify residues playing a critical role in mosquito-larvicidal activity. Larvicidal activity assays revealed that only F149A and Y150A mutants exhibited a total loss of toxicity. The in vitro interaction assays demonstrated that all BinB mutants are able to interact with BinA. Immunohistochemistry analysis revealed that only the Y150A mutant was unable to bind to the larval midgut, suggesting an important role of this residue in receptor binding of the BinB subunit. Conservative aromatic substitutions at F149 and Y150 resulted in full recovery of larvicidal activity, indicating that the aromaticity of F149 and Y150 is a key determinant of larvicidal activity, possibly playing a key role in the membrane interaction and receptor binding.