Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados. Cinvestav-Irapuato, Apartado postal 692, 36500, Irapuato, GTO., México.
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), 62250, Cuernavaca, MOR, México.
Antonie Van Leeuwenhoek. 2021 Nov;114(11):1759-1770. doi: 10.1007/s10482-021-01635-2. Epub 2021 Sep 7.
Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.
苏云金芽孢杆菌是农业中防治不同害虫和疾病传播媒介最成功的微生物杀虫剂。其活性主要归因于其孢子形成阶段表达的 Cry 蛋白。然而，这些蛋白并非苏云金芽孢杆菌所独有。一些 cry 基因已在其他芽孢杆菌种中发现，甚至在其他属中也有发现。在这项工作中，我们在 223 株无晶体芽孢杆菌菌株中搜索了 cry 基因。从这些菌株中获得了 13 个扩增子，克隆并测序；然而，只有 6 个扩增子对 cry 样基因呈阳性反应，且这 6 个分离株被证明是同一菌株。我们报告了一株异常的蜡状芽孢杆菌（LBIC-004）的特征，该菌株在孢子形成阶段无法形成蛋白内含物。LBIC-004 与蜡状芽孢杆菌使用 16S rRNA、gyrB 和 hag 基因的序列具有高度的同一性；此外，还获得了该菌株独特的质粒图谱。鉴定出一个 1953-bp 的 cry 基因，编码一个 651 个氨基酸的蛋白，分子量为 74.9 kDa。该蛋白显示出一种预测的三结构域结构，与所有 Cry 蛋白相似。然而，该蛋白的氨基酸序列与其最高同源物（Cry8Ca1 蛋白）的同源性仅为 41%，表明该 cry 样基因具有独特性。该基因被克隆并转移到一株无晶体的突变苏云金芽孢杆菌菌株中，用于对秀丽隐杆线虫、埃及伊蚊、烟青虫和 Phyllophaga sp.的生物测定。重组菌株未形成晶体，对测试的物种也没有毒性。
