【作者】 刘鹏；

【导师】 马培艳； Etienne Maisonneuve；

【作者基本信息】 武汉理工大学 ， 化学工程与技术， 2023， 硕士

【摘要】 在大肠杆菌(Escherichia Coli)的严紧反应中,核心信号分子(p)ppGpp的合成与水解平衡不仅与单一功能合成酶Rel A有关,而且依赖于具有水解和合成活性的双功能酶SpoT。蛋白质YmgB与酶SpoT比例的精确调控决定了SpoT的酶活性,因此细菌能够通过调控ymg B基因在细胞内的表达水平来控制SpoT依赖途径的(p)ppGpp累积。一直以来,YmgB都被认为是耐酸性和生物膜形成的调节剂,但其在严紧反应中的作用并未得到定论。已有充分证据表明,(p)ppGpp在细菌的持续性感染与多重抗药性中发挥着重要作用,对ymg B基因表达的调控可以构成细胞内(p)ppGpp水平控制的关键点。为了在此基础上研究ymgB基因表达的分子调节机制,更好的理解其在SpoT依赖的严紧反应途径中发挥的作用,本工作中通过构建lacZ融合质粒开发了一种基因筛选方案,用于识别调节ymgB表达的新元素。由于ymgB是ycgZ、ymgA、ymgB、ymgC四个基因组成的操纵子上的第三个基因,ymg B的表达受到位于ycgZ前的启动子PycgZ控制。为了检验在操纵子内是否存在其他ymgB启动子,本工作中选取了操纵子上三个基因片段F1(373bp)、F2(652bp)、F3(1028 bp)。通过融合三个片段与lacZ基因,共设计了6种不同的融合质粒。将其中最长片段F3组成的翻译融合质粒用于遗传筛选,共发现6个基因(gltD、soxS、yjbF、yafT、viaA和potD)蛋白产物的过量产生,促进了ymgB的表达。随后利用所有融合质粒对ymgB的表达进行分析,发现这些调节过程都发生在转录水平。并且发现在ymgB的5’端上游298个碱基中还存在着一个目前尚未确定的新启动子。该启动子上的表达受到CRP蛋白调控,且受到DNA结合转录调节蛋白SoxS,四种膜相关蛋白(YjbF,YafT,ViaA,PotD)以及葡萄糖的调控。其中膜相关蛋白的调控不通过CpxAR系统,葡萄糖的调控也不依赖CRP蛋白,说明该启动子受到多种不同转录调节因子的调控。另外从PycgZ开始的表达过程还受到一种谷氨酸合酶GltD的影响。该调控过程不依赖NtrBC双组分系统与σ因子RpoS,但在ntrB基因删除时却会导致显著的细胞活力下降,说明YmgB很可能在氮饥饿响应中发挥重要作用。总体来说,本工作构建了一种有效且易于拓展的活体细胞内基因调控分析方法,展现了两个启动子上对不同刺激的响应,为YmgB在严紧反应中发挥的作用展现了更多可能性。更多还原

【Abstract】 In the stringent response of Escherichia Coli,the synthesis and hydrolysis balance of the core signal molecule(p)ppGpp are not only related to the mono-function synthetase Rel A,but also depend on the bifunctional enzyme Spo T with hydrolysis and synthesis activities.Tight regulation of the Ymg B/Spo T ratio determines the enzymatic activities of Spo T,thus bacteria can control the(p)ppGpp accumulation in the Spo Tdependent pathways by regulating the intracellular expression level of ymgB gene.Ymg B has always been considered as a regulator of acid resistance and biofilm formation,but its role in stringent response has not been determined.Nowadays,there is sufficient evidence that(p)ppGpp plays an important role in persistent infection and multidrug tolerance of bacteria,and the regulation of ymgB gene expression can constitute the key point of controlling the intracellular(p)ppGpp level.To study the molecular regulation mechanism of ymgB gene expression and better understand its role in the Spo T-dependent stringent response pathway,a genetic screen was developed by constructing lac Z fusion plasmids to identify new elements that regulate ymgB expression.Overall,this work constructs an effective and easily scalable method for in vivo gene regulation analysis,demonstrating the response to different stimuli on two promoters,Since ymgB is the third gene on the operon composed of ycgZ,ymgA,ymgB,and ymgC,the expression of ymgB is controlled by the operon promoter Pycg Z which is located before ycg Z.In order to test whether there is another ymgB promoter in the operon,3 gene fragments F1(373bp),F2(652bp),and F3(1028bp)on the operon were selected in this work.By fusing these 3 fragments with the lacZ gene,6 different fusion plasmids were designed to analyze the gene expression of ymgB.The translation fusion plasmid composed of the longest fragment F3 was used in the genetic screen.It was found that the overproduction of proteins expressed by 6 genes(gltD,soxS,yjbF,yafT,viaA,and potD)promoted the expression of ymgB.All fusion plasmids were then used to analyze the expression of ymgB.The results suggest that those direct or indirect regulations occur at the transcriptional level.The experimental results also suggest the presence of a currently unidentified promoter within the 5’-upstream 298 bases of ymgB.The expression on this promoter is regulated by CRP proteins,the DNA-binding transcriptional regulator protein SoxS,four membrane-associated proteins(YjbF,YafT,ViaA,PotD),and glucose.Among them,the regulation of membrane-associated proteins does not pass through the CpxAR system,and the regulation of glucose does not depend on CRP proteins,suggesting that this promoter is regulated by a variety of different transcriptional regulators.In addition,the expression process starting from PycgZ is influenced by a glutamate synthase GltD.This regulatory process is not dependent on the NtrBC two-component system and the σ-factor RpoS.But it leads to a significant decrease in cell viability when the ntrB gene is deleted,suggesting that YmgB is likely to play an important role in the nitrogen starvation response.Overall,this work not only constructs an effective and easily scalable method for gene regulation analysis in vivo,but also demonstrates the response to different stimuli on two promoters,while showing more possibilities for the role played by YmgB in stringent response.更多还原