Lereclus D, Agaisse H, Gominet M, Chaufaux J
Unité de Biochimie Microbienne, URA 1300, Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France.
Biotechnology (N Y). 1995 Jan;13(1):67-71. doi: 10.1038/nbt0195-67.
The spo0A gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spo0A mutant of B. subtilis. In this paper we describe the construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spo0A gene of Bt was cloned and identified by its ability to transform a B. subtilis spo0A mutant to prototrophy. Its nucleotide sequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a disrupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spo0A mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulated toxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spo0A mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.
枯草芽孢杆菌的spo0A基因编码参与芽孢形成起始过程的关键因子。先前研究表明，编码对鞘翅目幼虫有活性的毒素的苏云金芽孢杆菌（Bt）cryIIIA基因，在枯草芽孢杆菌的spo0A突变体中过表达。在本文中，我们描述了Bt spo0A突变体菌株的构建及其在生产杀虫晶体蛋白中的应用。通过将枯草芽孢杆菌spo0A突变体转化为原养型的能力，克隆并鉴定了Bt的spo0A基因。其核苷酸序列与枯草芽孢杆菌基因同源。在Bt基因组中用一个破坏的拷贝替换spo0A基因，得到一个无法起始芽孢形成的Spo-菌株。当cryIIIA基因克隆到Bt spo0A突变体中时，产生并积累了大量毒素，形成一个大的晶体包涵体，该包涵体仍包裹在空细胞内。这些包裹的毒素对鞘翅目幼虫具有高度活性。我们预计，cryIIIA表达系统和Bt spo0A突变体将为生产稳定且环境安全的新型Bt基生物农药制剂提供一个便利的方法。
