Identification and characterization of new sRNA/mRNA targets of CsrA in Bacillus subtilis

枯草芽孢杆菌是最重要的非致病性革兰氏阳性模式菌，可向生长培养基中大量分泌蛋白质，因此可用于多种酶的工业生产。与革兰氏阴性模式生物大肠杆菌相反，枯草芽孢杆菌具有天然的能力，可以在营养压力下产生孢子，从而在极端条件下生存。此外，由于它与炭疽芽胞杆菌、蜡样芽胞杆菌等致病性芽胞杆菌有亲缘关系，因此它也可以作为调节这些致病性芽胞杆菌代谢反应或应激的模式生物。在革兰氏阴性菌中，RNA伴侣蛋白Hfq在sRNA稳定性和sRNA/靶mRNA相互作用中起重要作用，但在枯草芽孢杆菌和大多数革兰氏阳性菌中未发现这种作用。直到最近，还没有RNA伴侣被证明可以影响枯草芽孢杆菌中反式编码的sRNAs与其靶RNA之间的碱基配对。2019年，我们在枯草芽孢杆菌中发现并详细研究了广泛存在的RNA伴侣蛋白CsrA在这方面的新功能，即促进反式编码的sRNA SR1与其靶标ahrC mRNA之间的复合物形成。我们假设这种功能并不局限于SR1/ahrC mRNA调控系统。该项目的两个主要目的是：1)鉴定枯草芽孢杆菌中SR1的新的依赖于CsrA的mRNA靶点，并进一步表征CsrA在这些系统中的作用；2)采用一种全局方法鉴定枯草芽孢杆菌中所有与CsrA结合的rna。实验工作将分为以下三个部分，可以并行进行：1)鉴定SR1依赖于CsrA的新的mRNA靶点；阐明CsrA对这些mRNA的作用机制及在这些系统中的作用表征2)探讨CsrA对srmrna功能的可能作用13)通过CLIP Seq鉴定枯草芽孢杆菌CsrA的所有RNA结合伙伴，并通过EMSA和DRaCALA验证CsrA与选定RNA的结合

B. subtilis is the most important non-pathogenic Gram-positive model species that can secrete proteins in large quantities into the growth medium and is, therefore, used for the industrial production of a number of enzymes. In contrast to the Gram-negative model organism E. coli, B. subtilis is naturally competent and can sporulate in response to nutritional stress to survive extreme conditions. Moreover, as it is related to pathogenic Bacilli as e.g. B. anthracis or B. cereus, it can be used as a model organism for the regulation of metabolic responses or stress of such pathogenic bacteria as well. Whereas the RNA chaperone Hfq plays an important role for sRNA stability and sRNA/target mRNA interactions in Gram-negative bacteria, no such role has been found in B. subtilis and the majority of other Gram-positive bacteria. Until recently no RNA chaperones were known that were demonstrated experimentally to impact base-pairing between trans-encoded sRNAs and their target RNAs in Bacillus subtilis.In 2019, we have discovered and investigated in detail a new function for the widespread RNA chaperone CsrA in this respect, namely the promotion of complex formation between the trans-encoded sRNA SR1 and its target, ahrC mRNA, in Bacillus subtilis. We assume that this function is not confined to the SR1/ahrC mRNA regulatory system. The two major aims of this project are 1) to identify new CsrA-dependent mRNA targets of SR1 in B. subtilis and to further characterize the role of CsrA in these systems and 2) to employ a global approach to identify all CsrA-bound RNAs in B. subtilis.The experimental work will be divided into the following three parts which might be performed in parallel1) Identification of new mRNA targets of SR1 that depend on CsrA, elucidation of the mechanism of action of CsrA on these mRNAs and characterization of the role of CsrA in these systems2) Investigation of a possible role of CsrA for the mRNA function of SR13) Identification of all RNA binding partners of B. subtilis CsrA by CLIP Seq and verification of CsrA binding of selected RNAs by EMSA and DRaCALA