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

点击翻译按钮获取中文摘要

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.