Regulation of Bacterial Two-Component Signaling by Small Membrane Proteins

描述(申请人提供)：双组分系统是细菌用来感知和响应环境的主要信号转导模式之一。这些回路在使细菌能够适应不同的生长条件、控制发育程序和启动致病生活方式方面发挥着关键作用。典型的双组分系统由两个蛋白质组成，上游的组氨酸激酶通常参与信号检测，下游的反应调节器控制电路输出。信息流是通过将一个磷酸化基团从组氨酸激酶转移到反应调节器来实现的。然而，双组分系统可能会偏离这一简单的体系结构，并具有额外的蛋白质组分或更复杂的磷转移路径。在以前的工作中，我们发现一种由47个氨基酸组成的小分子膜蛋白MgrB可以抑制PhoQ的活性。由于MgrB的表达是由Phop激活的，因此该蛋白在PhoQ/Phop电路中作为负反馈回路的一部分发挥作用。PhoQ还受到另一种小分子膜蛋白SafA的刺激，SafA的表达受酸响应的EvgS/EVGA双组分系统控制。MgrB和SafA是最近发现的一组调节组氨酸激酶功能的小型疏水蛋白之一。在这个提议中，我们将结合荧光显微镜来跟踪单细胞中的电路行为，并结合遗传分析和建模来探索MgrB和SafA在PhoQ/Phop信号转导中的作用。我们将确定这些蛋白质在镁、抗菌肽和pH刺激下对PhoQ/Phop信号的动力学和输入输出行为的影响，比较自然大肠杆菌分离株的低pH响应行为，表征通过MgrB调节PhoQ/Phop电路的新输入信号，并研究与MgrB-PhoQ-Phop途径调节失调相关的生长缺陷。

DESCRIPTION (provided by applicant): Two-component systems are one of the primary modes of signal transduction that bacteria use to sense and respond to their environment. These circuits play a key role in enabling bacteria to adapt to diverse growth conditions, control developmental programs, and initiate pathogenic lifestyles. The prototypical two-component system consists of two-proteins, an upstream histidine kinase that is usually involved in signal detection, and a downstream response regulator, which controls the circuit output. Information flow occurs by transfer of a phosphoryl group from the histidine kinase to the response regulator. Two-component systems can deviate from this simple architecture, however, and have additional protein components or more complex phosphotransfer paths. In previous work we discovered that a small 47 amino acid membrane protein, MgrB, inhibits PhoQ activity. Since MgrB expression is activated by PhoP, the protein functions as part of a negative feedback loop in the PhoQ/PhoP circuit. PhoQ is also stimulated by SafA, another small membrane protein, whose expression is controlled by the acid-responsive EvgS/EvgA two component systems. MgrB and SafA are among a group of recently discovered small hydrophobic proteins that modulate histidine kinase function. In this proposal we will combine fluorescence microscopy to follow circuit behavior in single cells, with genetic analysis and modeling to explore the role of MgrB and SafA in PhoQ/PhoP signaling. We will determine the effect of these proteins on the dynamics and input-output behavior of PhoQ/PhoP signaling for stimulation with magnesium, antimicrobial peptides and pH, compare the behavior of the low pH response among natural E. coli isolates, characterize new input signals that modulate the PhoQ/PhoP circuit through MgrB, and study the growth defect associated with dysregulation of the MgrB-PhoQ-PhoP pathway.