Cyclic di-GMP mediated mechanisms on the “gearbox” of the bacterial flagellum

细菌的大部分时间都是作为细胞聚集体（称为生物膜），偶尔游动以殖民新的环境。为了游到这些新的栖息地，大多数细菌使用鞭毛，细胞壁嵌入旋转马达，旋转螺旋丝作为分子推进器。至关重要的是，鞭毛在形成生物膜时必须在新的栖息地中失活。初步研究表明，马达失活是环二聚鸟苷一磷酸（cdG），生物膜形成的中央调节器的行动的结果。在模式生物Caulobacter crescentus中，最近已经鉴定了多种未表征的cdG结合蛋白，其结合鞭毛马达的功能核心，“C环”，位于扭矩产生、旋转方向切换和信号响应的中心的多蛋白质胞质环。虽然这些cdG结合蛋白已被证明可以使马达失活，但其潜在的分子机制仍然未知。我建议使用电子冷冻断层扫描直接可视化的构象的C。crescentus C-环原位结合时，每个cdG信号蛋白，以了解其作用机制。为了鉴定不同信号传导蛋白的作用，将对除相关蛋白之外的所有蛋白的细菌菌株进行成像;随后，将在高cdG背景下过表达所讨论的蛋白以使结合饱和。为了解释观测结果，我还将对被设计成锁定在关键构象状态的C环突变体进行成像。这项工作将与Urs Jenal实验室合作进行，Urs Jenal是cdG作用的专家，也是这些cdG结合蛋白的发现者。结合，我预计结果将提供关键的见解如何cdG调节过渡到生物膜形成。

Bacteria spend the majority of their time as cellular aggregates called biofilms, sporadically swimming to colonize new environments. To swim to these new habitats most bacteria use flagella, cell wall-embedded rotary motors that spin a helical filament to function as a molecular propeller. Crucially, flagella must be deactivated in the new habitat when forming a biofilm. Preliminary studies suggest that motor deactivation is the result of action of cyclic dimeric guanosine monophosphate (cdG), a central regulator of biofilm formation. In the model organism Caulobacter crescentus, multiple uncharacterized cdG binding proteins have recently been identified that bind the functional core of the flagellar motor, the “C-ring”, a multiprotein cytoplasmic ring that lies at the heart of torque generation, rotational direction switching, and signal response. Although these cdG-binding proteins have been shown to deactivate the motor, the underlying molecular mechanism remains unknown. I propose using electron cryo-tomography to directly visualize the conformation of the C. crescentus C-ring in situ when binding each cdG signaling protein to understand the mechanism of their action. To identify the effects of different signaling proteins, bacterial strains gutted of all but the relevant protein will be imaged; subsequently, the protein in question will be overexpressed in a high cdG background to saturate binding. To interpret observations I will also image C-ring mutants engineered to be locked in key conformational states. Work will be conducted in collaboration with the lab of Urs Jenal, experts in cdG action and discoverers of these cdG-binding proteins. Combined, I anticipate results will provide key insights into how cdG regulates the transition to biofilm formation.