Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus thuringiensis
Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus thuringiensis
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全基因组筛选揭示苏云金芽孢杆菌抗生素杀虫剂的遗传决定因素
DOI：
10.1074/jbc.m110.148387
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发表时间：
2010-12-01
影响因子：
4.8
通讯作者：
Sun, Ming
中科院分区：
文献类型：
--
作者：
Liu, Xiao-Yan;Ruan, Li-Fang;Sun, Ming
苏云金素是苏云金芽孢杆菌中的一种耐热次级代谢产物，对多种昆虫具有杀虫活性。到目前为止，苏云金素生产的调控机制和遗传决定因素仍不清楚。在这里，我们成功地使用异源表达引导筛选大肠杆菌-苏云金芽孢杆菌穿梭细菌人工染色体文库，克隆完整的苏云金素合成（thu）集群。然后将该簇定位在菌株CT-43中带有杀虫剂晶体蛋白基因cry 1Ba的110-kb内源质粒上。并对该质粒进行了间接克隆和测序，命名为pBMB 0558。基于GenBank（TM）和KEGG数据库，对pBMB 0558上的基因功能进行了BLAST注释。pBMB 0558上的基因可分为三个功能模块：苏云金素合成簇、IV型分泌系统样模块和移动的遗传元件。采用高效液相色谱-质谱联用技术、大气压离子阱电离技术和飞行时间技术对苏云金素的生物合成中间体进行了检测。体内和体外活性测定证明thuE基因负责苏云金素磷酸化的最后一步。推导并阐明了苏云金素的生物合成途径。我们建议，苏云金素是一个腺嘌呤核苷寡糖，而不是一个腺嘌呤核苷酸类似物，传统上认为，基于预测的关键酶，糖基转移酶（ThuF）和胞外多糖聚合蛋白（Thu 1）的功能。
Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli-Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBank (TM) and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).