Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, Thailand.
FEMS Microbiol Lett. 2010 Feb;303(1):84-91. doi: 10.1111/j.1574-6968.2009.01865.x. Epub 2009 Nov 23.
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: (111)YLD(113)-->(111)AAA(113), (115)NNH(117)-->(115)AAA(117), (143)GEQ(145)-->(143)AAA(145), (147)FQFY(150)-->(147)AAAA(150), N114A and F146A. Only the replacements at (147)FQFY(150) 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.
球形芽孢杆菌产生一种由 BinB 和 BinA 亚基组成的杀蚊幼虫双毒素。BinA 对毒性很重要，而 BinB 则作为一种特定的受体结合成分起作用。为了研究仅存在于 BinB 中的两个区域的功能意义，最初引入了四个阻断突变和两个单突变：（111）YLD（113）-->（111）AAA（113），（115）NNH（117）-->（115）AAA（117），（143）GEQ（145）-->（143）AAA（145），（147）FQFY（150）-->（147）AAAA（150），N114A 和 F146A。只有（147）FQFY（150）的替换导致对库蚊幼虫的毒性完全丧失。进一步在该区域引入单点丙氨酸替换，F147A、Q148A、F149A 和 Y150A，以鉴定在杀蚊幼虫活性中起关键作用的残基。杀幼虫活性测定表明，只有 F149A 和 Y150A 突变体表现出完全丧失毒性。体外相互作用测定表明，所有 BinB 突变体都能够与 BinA 相互作用。免疫组织化学分析表明，只有 Y150A 突变体不能与幼虫中肠结合，表明该残基在 BinB 亚基的受体结合中起重要作用。F149 和 Y150 的保守芳香族取代导致杀幼虫活性完全恢复，表明 F149 和 Y150 的芳香性是杀幼虫活性的关键决定因素，可能在膜相互作用和受体结合中起关键作用。
