苏云金芽胞杆菌clpP基因缺失对碱刺激敏感性的影响

Effect of clpP disruption on alkaline sensitivity of Bacillus thuringiensis

【目的】比较苏云金芽胞杆菌与枯草芽胞杆菌在碱性培养条件下生长情况,明确clpP基因在碱刺激条件下的作用。【方法】采用同源重组技术敲除苏云金芽胞杆菌HD73菌株clpP基因,通过在不同pH下生长曲线的测定明确了clpP基因缺失突变体对碱性环境的敏感性,测定clpP基因的缺失对芽胞形成率、芽胞萌发效率和盐胁迫的影响。【结果】苏云金芽胞杆菌在碱刺激后,当培养基pH值为8.9-9.1时可以恢复生长,而枯草芽胞杆菌在pH值为8.2-8.4时可以恢复生长,说明苏云金芽胞杆菌对碱性环境适应能力更强,这有助于作为病原菌的Bt适应昆虫中肠的碱性环境。clpP基因缺失对芽胞形成率和萌发效率没有明显的影响。在将培养基中NaOH终浓度调节至30 mmol/L NaOH时,clpP基因缺失突变体的生长较出发菌株慢。说明ClpP在苏云金芽胞杆菌对碱性环境的适应过程中具有重要作用。

[Objective] We compared alkaline tolerance between Bacillus thuringiensis（Bt） and B.subtilis（Bs） and to determine the effect on growth when the clpP gene encoding caseinolytic protease is disrupted after alkaline shock.[Methods] B.thuringiensis HD73 mutant with the deletion of clpP gene was constructed by homologous recombination.The effects of clpP deletion on the growth after alkaline shock,sporulation and germination were analyzed.[Results] Bt can recover growth from alkaline shock when the medium pH was between 8.9 and 9.1 whereas that was between 8.2 and 8.4 for Bs.Bt tolerated alkaline more than Bs,leading Bt to adapt in alkaline environment of the midgut as a pathogen of insect.Deletion of clpP gene had no influence on sporulation and germination.The clpP mutant grew slower than Bt HD73 in the LB medium in addition to NaOH of 30 mmol/L.It indicates that the ClpP plays an important role in the alkaline tolerance of Bt strain.