Bacilysin在解淀粉芽孢杆菌中产孢功能与机制

Bacilysin, a dipeptide antibiotic, is produced by nonribosomal synthesized pathway. Bacilysin is active against a broad range of microorganism and the antimicrobial action mainly relies on its transport into host cells to interfere with the synthesis of glucosamine and consequently with cell wall formation. The structure of bacilysin likes quorum sensing signaling molecule Phr, which led to a assumption whether bacilysin could be transfered into cell again and have regulatory function on itself. Moreover, there is no study about this. Our previous studies demonstrated that bacilysin could be transfered into cytoplasm and increase sporulation-related genes and proteins expression levels, thus inducing sporulation. Based on current research, the objective of this study is to investigate the interacting proteins of bacilysin, deeply analyze their interaction between key proteins during sporulation and explore the mechanism of bacilysin regulating sporulation in Bacillus amyloliquefaciens. Results of this project will provide a new perpective to the biological function of secondary metabolites and scientific basis for strategy of highly efficient Bacillus based bioinoculants.

Bacilysin是芽孢杆菌通过非糖体途径合成的二肽类次级代谢产物，它被分泌到胞外后进入受体微生物体内，作用于细胞壁导致细胞溶解死亡从而具有广谱的抗菌功能。Bacilysin与群集感应信号分子寡肽Phr结构的相似性引得我们猜想bacilysin是否同样可以重新被运回到胞内，对自身细胞产生调控作用，并且该方面目前没有相关研究。申请人前期研究发现bacilysin能够被转运回芽孢杆菌细胞内，诱导产孢相关基因和蛋白上调表达，调控芽孢的形成。在此基础上，本项目将进一步鉴定bacilysin胞内互作蛋白，明确bacilysin及互作蛋白与产孢途径关键元件的互作关系，解析bacilysin调控产孢的具体分子机制。预期结果将为次级代谢产物的生物学功能开辟新观点，并为高效稳定的芽孢杆菌制剂的研发提供科学依据。

Bacilysin是芽胞杆菌通过非糖体途径合成的二肽类次级代谢产物，它被分泌到胞外后进入受体微生物体内，作用于细胞壁导致细胞溶解死亡从而具有广谱的抗菌功能。Bacilysin与群集感应信号分子寡肽Phr结构的相似性引得我们猜想bacilysin是否同样可以重新被运回到胞内，对自身细胞产生调控作用，并且该方面目前没有相关研究。本项目首先通过定点突变及过表达手段明确了bacilysin能够调控芽胞杆菌芽胞的形成过程。蛋白质组学分析表明与野生型相比，敲除突变体菌株下调表达蛋白32个；过表达菌株上调表达蛋白21个；两者相同差异表达蛋白共10个，其中包括芽胞形成起始的关键蛋白Spo0A和芽胞衣合成蛋白CotE。qRT-PCR结果显示突变bacilysin合成基因后，spo0A和cotE蛋白编码基因的表达量显著下调。EMSA验证结果表明Bacilysin不能与这2个基因启动子区域结合，说明bacilysin可能通过调控其它产胞调控因子从而间接影响。免疫共沉淀技术鉴定到与bacilysin有互作关系的蛋白共有16个，其中具有产胞相关功能的有2个，分别为芽胞形成后期蛋白SpoVT和SpoIIE；Pull down体外实验验证表明bacilysin与这2个蛋白在体外均不能直接结合，可能存在其它调控因子来传递。综述所述，本项目鉴定到了bacilysin胞内的互作蛋白，明确了bacilysin与产胞途径关键元件的具体互作关系，解析了bacilysin调控产胞的分子机制，为次级代谢产物的生物学功能开辟新观点，并为高效稳定的芽孢杆菌制剂的研发提供科学依据。