Spatiotemporal control of c-di-GMP signaling in Shewanella via phosphodiesterase PdeB

细菌细胞具有高度的细胞内组织，这需要适当的时空调节模式。C-di-GMP是一种重要的细胞内第二信使分子，参与多种细菌的多种过程调控。大多数细菌物种都含有过多的与c-di-GMP代谢有关的蛋白质和各种潜在的c-d-GMP结合蛋白，这就提出了如何激发适当的时空调节反应的问题。在以前的研究中，我们已经探索了希瓦氏杆菌属的伽马蛋白细菌将鞭毛系统和趋化系统招募到适当的细胞极点的机制。最近，我们发现了一种降解c-di-GMP的磷酸二酯酶PdeB，它对鞭毛马达的正常功能至关重要。依赖于极性标记HuBP，PdeB被招募到有鞭毛的细胞极点。我们的初步数据有力地表明，PdeB以一种未知的机制影响鞭毛电机的性能，推测是通过其具有未知结构的周质结构域感知到信号。在这个拟议的项目中，我们将努力阐明信号输入以及PdeB影响鞭毛运动的机制，我们的目标是了解磷酸二酯酶的时空定位如何具体影响细胞过程。为此，我们将异源制备和纯化PdeB的结构域以确定其活性，并将迄今尚未确定的假定信号感知结构域结晶以确定信号感知的结构和潜在机制。蛋白质下拉和相互作用研究将确定介导PdeB的极性招募、信号感知和表型输出的潜在因素。为了确定c-di-GMP水平影响鞭毛功能的机制，我们将进行与鞭毛和趋化系统的主要成分的直接结合研究。同时，我们将使用全球突变方法和c-di-GMP结合蛋白捕获试验来揭示c-di-GMP调控网络中与运动性和进一步的细胞过程有关的因素。通过荧光显微镜方法，将确定PdeB的定位模式。在一种互补的方法中，我们将开发一种基因系统，该系统将允许将c-d-GMP降解或合成活性靶向到特定的细胞间隔。该系统将用于具体研究局部活动对c-di-GMP分布的影响和细胞过程的调节。

Bacterial cells possess a high degree of intracellular organization, which require appropriate spatiotemporal regulational patterns. c-di-GMP has emerged as important intracellular second messenger molecule which is involved in regulation of a wide array of processes in numerous bacterial species. Most bacterial species harbor a plethora of proteins involved in c-di-GMP metabolism and various potential c-d-GMP-binding proteins, raising the question of how the appropriate spatiotemporal regulatory responses are elicited. In previous studies we have explored mechanisms by which gammaproteobacteria of the genus Shewanella recruit the flagellar and chemotaxis systems to the appropriate cell pole. Recently, we have identified a c-di-GMP-degrading phosphodiesterase PdeB which is crucial for proper function of the flagellar motor. PdeB is recruited to the flagellated cell pole in dependence of the polar marker HubP. Our preliminary data strongly indicate that PdeB affects the performance of the flagellar motor by an unknown mechanism, putatively upon perceiving a signal via its periplasmic domain with an unknown structure. Within this proposed project, we will take efforts to elucidate the signal input as well as the mechanism by which PdeB affects the flagellar motor, and we are aiming at understanding how spatiotemporal localization of a phosphodiesterase specifically affects cellular processes. To this end, we will heterologously produce and purify domains of PdeB to determine their activity, the hitherto uncharacterized putative signal-perceiving domain will be crystallized to identify structure and potential mechanism of signal perception. Protein pull-downs and interaction studies will identify potential factors mediating polar recruitment, signal perception, and phenotypic output of PdeB. To determine the mechanism by which c-di-GMP levels affect flagellar functions, we will perform direct binding studies with major components of the flagella and chemotaxis systems. In parallel, we will use a global mutagenesis approach and a c-di-GMP-binding protein capture assay to unveil factors of the c-di-GMP regulatory network with respect to motility and further cellular processes. By fluorescence microscopy approaches, the localization patterns of PdeB will be determined. In a complementary approach we will develop a genetic system that will allow targeting of c-d-GMP-degrading or -synthesizing activity to specific cell compartments. This system will be applied to specifically study the effect of local activity on the distribution of c-di-GMP and regulation of cellular processes.