Characterization of OmpR Gene Regulation

描述（由申请人提供）：两个组成部分的监管系统已经成为适应性反应的范例。最简单的系统由传感器和响应调节器组成。大肠杆菌中调节孔蛋白基因的双组分系统响应生长介质渗透压的变化。EnvZ，假定的渗透传感器被细胞内ATP磷酸化，然后磷酸化OmpR。在低渗透压下，外膜的主要孔蛋白是OmpF，在高渗透压下，OmpF转录被抑制，而ompC被激活。双组分系统密切参与许多不同病原体中毒力因子的协调表达，OmpR是一种重要的全球调节蛋白。在沙门氏菌中，OmpR位于调控级联的第一步，该级联开启了下游的双组分调控系统，并激活了全身性感染所需的第三型分泌系统的表达。在本应用中，需要验证的假设是EnvZ通过调节其磷酸酶活性来响应渗透信号，从而控制OmpR的浓度约为P。在之前的资助期，我们发现DNA结合刺激OmpR磷酸化。这一观察结果可能对信号传导具有重要的机制意义，它表明作为转录因子的反应调节因子在与DNA结合时可能被磷酸化。在第一个目标中，我们将尝试分离和表征这种EnvZ/OmpR/DNA复合物。EnvZ-GFP融合将用于检测完整细胞和球质体中的信号和转录，以确定外膜的作用（如果有的话）。一种OmpR约P去磷酸化试验已经开发出来，并将用于测试EnvZ在OmpR-P转换中的作用。在之前的资助期内，我们发现OmpR能够以头对头的方式与DNA结合，而不是之前提出的头对尾的方式。我们的新模型预测，识别螺旋实际上是螺旋2而不是螺旋3。我们将确定螺旋2和3在DNA识别中的作用，并验证OmpR可以以多个方向与DNA结合的假设。在目标三中，我们建议通过核磁共振来解决OmpR的全长结构，分离的N端结构域和c端结构域，磷酸化的N端结构域和与DNA结合的c端结构域的结构。

DESCRIPTION (provided by applicant): Two component regulatory systems have emerged as a paradigm for adaptive responses. The simplest systems consist of a sensor and a response regulator. The two-component system in E. coli that regulates the porin genes responds to changes in osmolarity of the growth medium. EnvZ, the presumed osmosensor is phosphorylated by intracellular ATP and then phosphorylates OmpR. At low osmolarity, the major porin in the outer membrane is OmpF and at higher osmolarity, ompF transcription is repressed and ompC is activated. Two-component systems are intimately involved in the coordinate expression of virulence factors in many different pathogens and OmpR is an important global regulatory protein. In Salmonella, OmpR lies at the first step of a regulatory cascade that turns on a downstream two-component regulatory system and activates expression of a type three secretion system required for systemic infection. In the present application, the hypothesis to be tested is that EnvZ controls the concentration of OmpR approximately P by adjusting its phosphatase activity in response to the osmotic signal. Prior to the previous funding period, we discovered that DNA binding stimulates OmpR phosphorylation. This observation may have important mechanistic implications for signaling and it suggests that response regulators that function as transcription factors may be phosphorylated while bound to the DNA. In the first aim, we will attempt to isolate and characterize such an EnvZ/OmpR/DNA complex. An EnvZ-GFP fusion will be employed to examine signaling and transcription in intact cells and in spheroplasts to determine the role (if any) of the outer membrane. An OmpR approximately P dephosphorylation assay has been developed and will be used to test the role of EnvZ in OmpR-P turnover. In the previous funding period, we discovered that OmpR is capable of binding to DNA in a head-to-head orientation rather than the previously proposed head-to-tail mode. Our new model predicts that the recognition helix is actually helix 2 rather than helix 3. We will determine the role of helices 2 and 3 in DNA recognition and test the hypothesis that OmpR can bind to DNA in more than one orientation. In aim three, we propose to solve the full-length structure of OmpR, the structures of the isolated N- and C-terminal domains, the phosphorylated N-terminal domain and the C-terminal domain bound to DNA by NMR.