Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.
Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.
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DOI：
10.1016/s0021-9258(18)45879-6
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发表时间：
1992-02
期刊：
影响因子：
--
通讯作者：
Dong Wu;A. Aronson
中科院分区：
文献类型：
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作者：
Dong Wu;A. Aronson
苏云金芽孢杆菌产生多种三角洲内毒素，与昆虫幼虫中肠的特定受体结合。在插入膜后，离子通量发生变化，最终导致渗透溶解。突变寡核苷酸被用来确定其中一种毒素中涉及特异性和毒性的区域。在迄今测得的所有毒素中，有一个区域高度保守，许多突变导致三个测试鳞翅目的毒性丧失。突变毒素失去了抑制依赖K(+)的氨基酸转运到幼虫中肠囊泡的能力，但不影响它们与野生型毒素竞争结合的能力。结果与毒素的这个两亲性螺旋区域是毒性所必需的一致。第二个诱变区与另一个潜在的两亲性螺旋的一部分重叠。只有两个残基Ala-92和Arg-93的突变导致两种鳞翅目幼虫失去毒性，但三分之一的幼虫仍有一些活性。A92D突变毒素与野生型毒素竞争结合敏感幼虫的中肠小泡，而不是不敏感幼虫的小泡。当这种突变转移到另外两个相关的原毒素基因上时，毒性也被降低。分析了每个残基的一些突变，选择性毒性损失与没有正电荷有关。尽管这些残基远离假定的特异性结构域，但1或两者都必须在毒素的特异性结合中发挥重要作用。
Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.