苏云金杆菌Bt4.0718不同时期比较转录组揭示芽胞和杀虫伴胞晶体形成机制

Comparative transcriptomics of Bacillus thuringiensis Bt4.0718 reveals the mechanisms of sporulation and parasporal crystal formation

摘要:

【目的】苏云金芽胞杆菌(Bacillus thuringiensis, Bt)在形成芽胞的过程中产生大量杀虫晶体蛋白(insecticidal crystal proteins, ICPs)，是目前应用最广泛、最安全的微生物杀虫剂的主要菌株资源。本研究旨在比较Bt 3个重要时期的转录组，进一步探究芽胞和杀虫伴胞晶体的形成机制，为高效工程菌的构建奠定理论基础。【方法】选取高毒力Bt4.0718菌株营养生长中期(T1-10 h)、芽胞形成前期(T2-20 h)、芽胞形成后期(T3-32 h)进行比较转录组分析，对代表性差异基因进行实时荧光定量PCR (real-time fluorescence quantitative PCR, qRT-PCR)验证、特定功能基因的敲除和表型分析验证。【结果】差异表达基因数量分别为2 147个(T2/T1)、1 861个(T3/T1)、1 708个(T3/T2)。T1时期，培养基中营养相对丰富，主要为芽胞和杀虫伴胞晶体形成做准备。芽胞形成重要调控基因kinA/D、spo0A/F、sigE高水平转录对菌体的生长发育具有重要作用，Cry1Ac、碳源、能源贮藏物聚-羟基丁酸(poly-hydroxybutyric acid, PHB)和羟基丁酮(acetoin)也已开始转录。芽胞和ICPs的大量形成在T2和T3时期，相关基因的转录水平T2比T3时期高。T2对比T3时期，芽胞核/衣/皮质(spore core/coat/cortex)、芽胞萌发受体蛋白(germination protein)和芽胞形成的II-VI阶段相关基因(spoII-spoVI)在T2时才开始大量转录，为3个时期的最高水平；相应的糖类、氨基酸、脂质代谢，能量、核酸、多肽代谢、次级产物生成和环境适应系统等复杂网络均表现出差异；另外，作为营养信号刺激性生理过程，双组分信号转导系统(two-component signal transduction system, TCS)和ABC转运系统在芽胞形成和ICPs的转录表达过程中发挥重要作用，转录水平具有显著差异。【结论】随着芽胞和杀虫伴胞晶体的形成，营养成分逐渐匮乏，sigB、sigW和sigM的高效表达有助维持细胞壁的稳定和对环境变化的抗性；小热激蛋白Hsp20和Hsp20B作为分子伴侣蛋白对维持胞内稳态也十分重要，T2、T3时期高水平转录可能有助于芽胞形成和ICPs表达。

Abstract:

[Objective] Bacillus thuringiensis (Bt), characterized by the massive production of insecticidal crystal proteins (ICPs) during sporulation, serves as the main strain resource for the commonly used and safe microbial insecticides. To further explore the mechanisms of sporulation and parasporal crystal formation and lay a theoretical foundation for the construction of efficient strains, we compared the transcriptomes of Bt at three important stages. [Methods] The transcriptomes of the hypervirulent strain Bt4.0718 at the middle vegetative growth stage (T1-10 h), the early sporulation stage (T2-20 h), and the late sporulation stage (T3-32 h) were compared. The representative differentially expressed genes (DEGs) were verified by real-time fluorescence quantitative PCR (qRT-PCR), and the phenotypes of the mutant strains with the knockout of specific functional genes were examined. [Results] The number of DEGs was 2 147 (T2/T1), 1 861 (T3/T1), and 1 708 (T3/T2), respectively. At T1, the medium was rich in nutrients, which served the sporulation and parasporal crystal formation. The high transcription levels of kinA/D, spo0A/F, and sigE regulating sporulation played a role in the growth and development of the cells. The transcription of Cry1Ac, poly-hydroxybutyric acid (PHB), and hydroxybutanone (acetoin) were started at this time. The substantial formation of ICPs and spores occurred at T2 and T3, and the transcript levels of the regulatory genes were higher at T2 than those at T3. The genes associated with spore core/coat/cortex, germination protein, and spoII–spoVI began to be transcribed in large amounts at T2, with the highest levels among the three stages. The corresponding complex networks of carbohydrate, amino acid, and lipid metabolism, energy, nucleic acid, and peptide metabolism, secondary metabolite production, and environmental adaptation showed differences. In addition, as the physiological processes stimulated by nutrient signals, the two-component signal transduction system (TCS) and ABC transport system played an essential role in the process of sporulation and ICP transcription and expression, and their transcription levels were significantly different. [Conclusion] With the production of ICPs and sporulation, nutrients are gradually consumed, and the high expression of sigB, sigW, and sigM contributed to the stability of cell wall and the resistance to environmental changes. Meanwhile, the small heat shock proteins Hsp20 and Hsp20B, as molecular chaperones, were also important for maintaining intracellular homeostasis and may facilitate the sporulation and ICP production.