Shivakumar A G, Gundling G J, Benson T A, Casuto D, Miller M F, Spear B B
J Bacteriol. 1986 Apr;166(1):194-204. doi: 10.1128/jb.166.1.194-204.1986.
Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.
苏云金芽孢杆菌库斯塔克亚种的总DNA用BglII酶切，并克隆到大肠杆菌质粒pUC9的BamHI位点。一种重组质粒pHBHE表达了一种对毛虫有毒的135,000道尔顿的蛋白质。然后将pHBHE的HincII - SmaI双酶切产物与经BglII酶切的质粒pBD64连接，并通过转化导入枯草芽孢杆菌。通过菌落杂交鉴定转化体，并通过Southern印迹杂交进行确认。从含有毒素基因插入片段（无论何种方向）的枯草芽孢杆菌克隆中检测到一种与苏云金芽孢杆菌晶体蛋白特异性抗体结合的135,000道尔顿蛋白质。克隆到pBD64载体两个耐药基因远端的PvuII位点的毒素基因插入片段也表达了一种135,000道尔顿的蛋白质。这些结果表明毒素基因是从其自身启动子转录的。对在不同生长阶段表达的蛋白质进行的蛋白质印迹分析表明，枯草芽孢杆菌中的晶体蛋白表达在营养生长阶段早期开始，而在苏云金芽孢杆菌中则与芽孢形成开始同时发生。当克隆基因转移到枯草芽孢杆菌的非芽孢形成菌株时，也表达了一种135,000道尔顿的蛋白质。这些结果表明枯草芽孢杆菌中的毒素基因表达与芽孢形成无关。另一个编码130,000至135,000道尔顿蛋白质的毒素基因从在λ1059中建立的苏云金芽孢杆菌基因文库中克隆到大肠杆菌中。然后将该基因亚克隆到枯草芽孢杆菌中。两个克隆的细胞提取物对毛虫都有毒性。电子显微镜研究表明，大肠杆菌中存在不规则的晶体包涵体，而枯草芽孢杆菌克隆中存在形态良好的双金字塔形晶体，类似于在苏云金芽孢杆菌中发现的晶体。
