Comprehensive analysis of transcriptional regulatory network in Escherichia coli

在本项目中，我们试图分析大肠杆菌中所有两个组成系统的整体功能。为此，我们分离了36个两个组成系统单独缺失的突变体。利用基因芯片和SELEX技术，我们可以识别或提示双组分系统识别的靶基因（或调节子）控制的顺式作用序列。此外，我们还可以识别出两个组分系统所感知的胁迫信号，我们发现胁迫可以分为代谢信号（碳、氮和与呼吸有关的化合物等），离子/金属胁迫（Fe、Cu、Mg、Zn、K和P等），以及多种胁迫（细胞表面胁迫、氧化胁迫和渗透胁迫等）。另外，我们还对所有的His激酶和反应调节因子进行了纯化，并进行了各种体外磷酸化实验。因此，我们可以确定非同源对的His激酶和反应调节剂，可以互相交叉（反式磷酸化）。有必要揭示的生理意义，建议在体内的crosstalk在future.In一个单独的分析，我们确定了两个组件系统，调节固定特定的σ因子（RpoS）。我们还鉴定了调控鞭毛表达的两个组分系统，在本项目中，制备了有用的资源，如所有两个组分系统的缺失突变体，所有两个组分基因的表达质粒，纯化的蛋白质，用于在大肠杆菌中进行双杂交分析的质粒等。

In this project, we tried to analyze the whole function of all two component systems in E coli For this, we isolated 36 two component deletion mutants in which each system is deleted individually. By using the microarray and the SELEX methods, we could identify or suggest the target genes (or regulon) controlled and cis-acting sequences recognized by two component systems. Moreover, we could identify the stress signals that were perceived by two component systems, and we revealed that the stresses could be categorized into metabolic' signals (carbon, nitrogen, and compound related to respiration etc), ion/metal stresses (Fe, Cu, Mg, Zn, K, and P etc.), and the diverse stresses (cell surface stress, oxidation stress, and osmotic stress, etc). We also suggested that there are signaling networks between two component systems.On the other hand, we purified all His kinases and response regulators and carried out every phosphorylation experiment in vitro. As a result, we could identify the non cognate pairs of His kinase and response regulators that could cross talk (trans phosphorylate) each other. It is necessary to reveal the physiological significance of the suggested cross talk in vivo in the future.In an individual analysis, we identified two component systems that regulate stationary specific sigma factor (RpoS). We also identified two component systems that regulate flagella expression.In this project, useful resources such as deletion mutants of all two component systems, expression plasmids of all two component genes, purified proteins, plasmids for two hybrid analysis in E coli etc. were prepared.