The nitrogen-related phosphotransferase system in Escherichia coli

氮相关的磷酸转移酶系统PTSNtr与催化细菌中碳水化合物吸收的典型PTS平行工作。PTSNtR在变形杆菌中高度保守，影响氮、碳同化和毒力等重要过程。然而，潜在的机制在很大程度上是未知的。PTSNtr由三种蛋白质组成：酶IntR(EINtr)，自磷酰化，并通过蛋白质NPR将磷酸基转移到酶IIANtr(EIIANtr)。最近，在大肠杆菌中发现了PTSNtr在K+吸收调节中的直接作用。特别是，EIIANtr结合并刺激组氨酸激酶KdpD的活性，进而激活高亲和力K+转运体KdpFABC的合成，以响应有限的K+浓度。初步数据表明，去磷酸化的EIIANtR与KdpD相互作用。在拟议的项目中，将探索这一调控的分子机制和生理意义。特别是，应该对参与相互作用的表面进行表征，以了解如何通过与辅助蛋白的相互作用来激活组氨酸激酶。其次，应明确EIIANtR的磷酸化状态对相互作用的作用，并识别触发磷酸化的信号。第三，EINtr包含一个GAF信号域，初始数据表明该域控制EIIANtr的调节输出。因此，应该确定与GAF结合并可能控制EINtr自动磷酸化的配体。最后，将探讨PTSNtr是否通过调节细胞内K+浓度而发挥其多效性作用，进而影响许多基因和酶的活性。

The nitrogen-related phosphotransferase system PTSNtr works in parallel to the canonical PTS that catalyzes uptake of carbohydrates in bacteria. PTSNtr is highly conserved in Proteobacteria and affects important processes such as nitrogen and carbon assimilation and virulence. The underlying mechanisms, however, are largely unknown. PTSNtr consists of three proteins: Enzyme INtr (EINtr) autophosphorylates and transfers phosphoryl-groups via protein NPr to enzyme IIANtr (EIIANtr). Recently, a direct role of PTSNtr in regulation of K+ uptake was revealed in Escherichia coli. In particular, EIIANtr binds and stimulates kinase activity of histidine kinase KdpD, which in turn activates synthesis of the high affinity K+ transporter KdpFABC in response to limiting K+ concentrations. Initial data suggest that dephosphorylated EIIANtr interacts with KdpD. In the proposed project, the molecular mechanism and the physiological meaning of this regulation shall be explored. In particular, the surfaces involved in interaction shall be characterized to learn how a histidine kinase can be activated by interaction with an accessory protein. Second, the role of the phosphorylation state of EIIANtr for interaction shall be clarified and the signals triggering phosphorylation shall be identified. Third, EINtr contains a GAF signaling domain and initial data suggest that this domain controls the regulatory output of EIIANtr. Hence, the ligand that binds to GAF and presumably controls EINtr autophosphorylation shall be identified. Finally, it shall be explored whether PTSNtr exerts its pleiotropic roles through modulation of the intracellular K+ concentration, which may in turn affect activities of numerous genes and enzymes.