Specificity of phosphatase activity and interaction of heme-responsive two-component systems in Corynebacterium glutamicum

双组分系统代表了一种普遍的细菌信号转导模式。新的信号通路的进化是基于基因复制事件和随后的分化，或通过水平基因转移获得新的系统。最近，我们描述了两个密切相关的双组分系统（TCS），它们协调控制了革兰氏阳性土壤细菌谷氨酸棒状杆菌对血红素作为替代铁源的利用和对高水平血红素的解毒。这些系统可能是最近基因复制事件的结果，但表现出特定的信号转导。HrrSA系统通过激活血红素加氧酶对血红素的利用至关重要，而ChrSA系统则需要通过激活假定的hrtBA输出器来应对高水平和有毒的血红素水平。最近的研究表明，这两种tcs都对血红素作为刺激作出反应，表现出高水平的串扰，但对其同源反应调节因子表现出特定的磷酸酶活性。由于这些独特的性质，TCSs HrrSA和ChrSA是研究传感器激酶磷酸酶特异性决定因素的理想模型，而磷酸酶特异性是确保密切相关系统通路绝缘的关键机制。在这项研究中，我们将使用随机和合理的方法来定义赋予磷酸酶反应特异性的界面。分子生物学研究将辅以组氨酸激酶和反应调节因子对的可溶性复合物或亚结构域的结构分析（与HHU dseldorf教授Georg Groth合作）。此外，我们将继续研究网络特性，包括蛋白质化学计量学和自动调节，特别关注ChrSA和HrrSA系统之间交叉磷酸化的生理相关性。除了经典的分子生物学方法外，我们将使用最先进的报告菌株活细胞成像技术，使用延时荧光显微镜来分析野生型和突变背景下信号转导的单细胞动力学。我们相信，我们之前的研究为计划中的实验提供了坚实的基础，并且从这个项目中获得的结果将为科学界普遍感兴趣的TCS信号转导和网络设计提供新的见解。

Two-component systems represent a prevalent mode of bacterial signal transduction. The evolution of novel signaling pathways is based on gene duplication events and subsequent divergence or on the acquirement of new systems by horizontal gene transfer. Recently, we described two closely related two-component systems (TCS) which coordinately control the utilization of heme as an alternative iron source and the detoxification of high heme levels in the Gram-positive soil bacterium Corynebacterium glutamicum. These systems are likely the result of a recent gene duplication event but exhibit specific signal transduction. Whereas the HrrSA system is crucial for the utilization of heme by activating the heme oxygenase, the ChrSA system is required to cope with high and toxic heme levels by activation of the putative hrtBA exporter. Recent studies revealed that both TCSs respond to heme as a stimulus, exhibit a high level of cross-talk but show specific phosphatase activity towards their cognate response regulator. Due to these unique properties, the TCSs HrrSA and ChrSA represent an ideal model to study the determinants of phosphatase specificity of sensor kinases, which is a key mechanism ensuring pathway insulation of closely related systems.In this study we will use random and rational approaches to define the interface conferring specificity to the phosphatase reaction. Molecular biology studies will be complemented by structural analysis of the soluble complexes or subdomains of histidine kinase and response regulator pairs (cooperation with Prof. Georg Groth, HHU Düsseldorf). Furthermore, we will continue our studies on the network properties including protein stoichiometry and autoregulation with a special focus on the physiological relevance of cross-phosphorylation between the ChrSA and HrrSA systems. Besides classical molecular biology approaches, we will apply state-of-the-art live cell imaging of reporter strains using time-lapse fluorescence microscopy to analyse single cell dynamics of signal transduction in wild type and mutant backgrounds. We are convinced that our previous studies provide a solid basis for the planned experiments and that the results obtained from this project will contribute novel insights into TCS signal transduction and network design which are of general interest for the scientific community.