c-di-GMP核糖开关Bc1 RNA介导的蜡样芽胞杆菌群细菌趋化性调节新机制

cyclic di-GMP is a universal and important bacterial second messenger. Its intracellular concentration is controlled through the antagonistic activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). c-di-GMP was found to regulate various important physiological processes in diverse bacteria by binding to a wide variety of protein effectors including kinases or phosphorylases, transcription factors, PilZ domain, MshEN domain, degenerate DGCs or PDEs, or RNA riboswitches. Riboswitches are structured RNAs typically located in the 5’-untranslated regions (5’-UTRs) of mRNAs to regulate expression of downstream genes in response to changing concentrations of their cognate ligands. Bc1 DNA is one of them, residing upstream of the open reading frame of a methyl-accepting chemotaxis protein (McpE), the structure of which is highly conserved in the Bacillus cerues group. MCP forms a complex with the sensor kinase CheA and triggers its autophosphorylation, which then phosphorylates downstream response regulator CheY. Phosphorylated CheY (CheY-P) subsequently binds to a flagellar motor to inhibit its rotation. On the other hand, CheR is another effector responsible for the methylation of MCPs to affect bacterial adaptation. The present study plans to focus on the chemotaxis regulatory mechanism of c-di-GMP/Bc1 RNA/McpE signaling pathway under both in vitro and in vivo conditions. We will use in vitro transcription termination and in-line probing assays to study the interaction between c-di-GMP and Bc1 RNA, as well as the conformation change of Bc1 RNA upon c-di-GMP binding. β-galactosidase and qRT-PCR assays will be used to illustrate the regulatory role of Bc1 RNA to the transcription of mcpE in response to different c-di-GMP levels. Besides McpE, B. thuringiensis BMB171 contains 12 different MCPs which might have cross-talk with McpE signaling pathway. To study the signaling mechanism of McpE, we will first knock out all of these 12 mcp genes (the mutant obtained was named ΔMCP), and then we will knock out Bc1 RNA and mcp gene in ΔMCP respectively, and label flagella with a red fluorescence protein to observe bacterial motility in real time by super-resolution fluorescence microscope to further elucidate the regulatory roles of c-di-GMP/Bc1 RNA/McpE signaling pathway in chemotaxis and motility. We will also study the function of McpE in response to different methylation levels by knocking-out or knocking-down cheR. The study shall reveal a new regulatory mode of c-di-GMP for bacterial chemotaxis and motility and provides an important RNA element for synthetic biology.

c-di-GMP是细菌中广泛存在的第二信使，通过与下游靶标蛋白或核糖开关的结合，调控细菌的多种重要生理功能。在一个甲基受体趋化蛋白McpE mRNA的5’-非翻译区存在一个c-di-GMP的核糖开关Bc1 RNA，这种结构仅存在于蜡样芽胞杆菌群中，且十分保守。本研究拟在苏云金芽胞杆菌BMB171内源性c-di-GMP浓度高、低突变株细胞内及体外研究Bc1对c-di-GMP的响应机制，筛选McpE特异性诱导剂，在敲除其他mcp基因基础上，分别敲除Bc1编码区、mcpE以及MCPs去甲基化蛋白CheR编码基因，通过实时观测荧光蛋白标记的细菌突变株运动规律，揭示c-di-GMP与受体Bc1作用，调控McpE的表达量及甲基化水平，从而影响响应调节子CheY磷酸化，进而调节鞭毛运动的信号通路。本研究不仅揭示一种新的趋化性调控方式，丰富c-di-GMP的调控网络，还为合成生物学提供重要的RNA元件。

蜡样芽胞杆菌群细菌中普遍存在一个核苷类第二信使分子c-di-GMP的RNA受体——c-di-GMP的核糖开关（将其命名为Bc1）。Bc1位于一个甲基受体趋化蛋白MCP (命名为McpE)编码基因转录本的5'-非翻译区，而MCPs与细菌的趋化与运动相关。目前蜡样芽胞杆菌群细菌中c-di-GMP 能否通过Bc1介导调控mcpE的表达而影响细菌运动的机理完全未知。.本研究以苏云金芽胞杆菌BMB171为例，探究了c-di-GMP对Bc1的调控模式。我们发现Bc1-mcpE上游存在活性较高的启动子，Bc1的结构比较复杂，其表达平台含有两个Rho因子非依赖性强终止子T1、T2以及SD序列。Bc1与c-di-GMP的结合方式并不像经典的c-di-GMP核糖开关（如Vc2）那样保守，Bc1主要在转录水平抑制mcpE的表达，该转录抑制与翻译起始无关；Bc1同样具有显著的翻译抑制调控作用。在低、中浓度c-di-GMP条件下主要是Bc1发挥作用允许mcpE表达，而在高浓度c-di-GMP条件下则是另外的调控因子促进mcpE的转录。Bc1、mcpE缺失和mcpE过表达并未显著影响细菌运动性，但mcpE过表达会使细菌生物被膜形成增加。我们发现，McpE胞外配体结合结构域LBD在体外能够与1.25、2.5 mM硫胺素焦磷酸（TPP）直接结合，初步验证TPP为其趋化物质。总之，蜡样芽胞杆菌群中c-di-GMP对核糖开关Bc1和下游基因mcpE的调控机制比目前报道的机制更加复杂，我们揭示了Bc1“双终止”的调控机制，是一种全新的c-di-GMP-核糖开关调控模式，这对于丰富核糖开关调控机制，为合成生物学研究挖掘新的RNA调控元件具有重要意义。

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