Sensory Mechanisms and Local Signaling in c-di-GMP-mediated Signal Transduction in Escherichia coli

细菌生物膜定植于不同的表面，并且对抗生素或宿主免疫系统具有高度抗性。在这些生物膜中，细胞嵌入在细胞外生物聚合物的自我产生的保护基质中。E.大肠杆菌是淀粉样卷曲纤维和胞外多糖纤维素，其合成受固定相sigma因子RpoS（<$S）和第二信使c-di-GMP控制。后者由二鸟苷酸环化酶（DGC，具有GGDEF结构域）产生，并由特异性磷酸二酯酶（PDE，具有EAL结构域）降解。在E. coliK-12中有12个是DGCs，13个是PDE。这些差异表达的DGC和PDE中的许多是膜相关的，并且它们的活性由N-末端感觉结构域控制。使用生物膜基质产生作为c-di-GMP靶标，其为遗传、分子生物学和生物化学分析提供方便的表型和读数，建议的项目旨在阐明c-di初级和次级水平信号感知和处理的分子机制，E.杆菌这包括（i）通过一类新型PDE的N-末端感觉输入结构域感测环境和细胞信号，以及（ii）通过新型和非正统的c-di-GMP效应器机制感测c-di-GMP。具体而言，将研究以下信号感知和c-di-GMP效应器机制：-通过六个E的周质环结构域中的二硫键形成和蛋白水解加工对细胞质酶活性的氧化还原控制。大肠杆菌PDE与CSS-EAL结构域架构-YbjK的分子结构，功能和目标，DeoR样转录因子，似乎结合c-di-GMP作为G-四链体-PdeL的转录调节，局部作用c-di-GMP结合和具有LuxR-EAL结构域架构的降解“触发PDE”和转录因子-推定的c-di-在orf 0317-pdeL操纵子的mRNA的基因间区域中的GMP核糖开关，其下调PdeL表达。将阐明这些分子过程在控制基质产生并由此产生生物膜的功能和结构的大的调节网络内的整合。在DGCs和PDE的控制，c-di-GMP传感效应器组件和本地c-di-GMP信号的感觉输入，该项目预计将作出重大贡献，实现SPP 1879的目标。此外，其结果将具有实际应用，例如在开发新的抗生物膜策略或合成生物学中。

Bacterial biofilms colonize diverse surfaces and are highly resistant against antibiotics or host immune systems. In these biofilms, cells are embedded in a self-produced protective matrix of extracellular biopolymers. Major biofilm matrix components in E. coli are amyloid curli fibres and the exopolysaccharide cellulose whose synthesis is under control of the stationary phase sigma factor RpoS (¿S) and the second messenger c-di-GMP. The latter is produced by diguanylate cyclases (DGC, with GGDEF domains) and is degraded by specific phosphodiesterases (PDE, with EAL domains). 25 of the 29 GGDEF/EAL domain proteins of E. coli K-12 are enzymatically active, with 12 being DGCs and 13 being PDEs. Many of these differentially expressed DGCs and PDEs are membrane-associated and their activities are controlled by N-terminal sensory domains.Using biofilm matrix production as a c-di-GMP target that provides for convenient phenotypes and readouts for genetic, molecular biological and biochemical analyses, the proposed project aims at clarifying the molecular mechanisms of signal sensing and processing at the primary and secondary levels of c-di-GMP signaling in E. coli. This includes (i) sensing environmental and cellular signals via the N-terminal sensory input domains of a novel class of PDEs, and (ii) sensing c-di-GMP via novel and unorthodox c-di-GMP effector mechanisms. In particular, the following signal perception and c-di-GMP effector mechanisms will be studied:- Redox control of cytoplasmic enzyme activity via disulfide bond formation and proteolytic processing in the periplasmic loop domain of the six E. coli PDEs with CSS-EAL domain architecture- Molecular structure, function and targets of YbjK, a DeoR-like transcription factor that seems to bind c-di-GMP as a G-quadruplex- Regulation of transcription by PdeL, a locally acting c-di-GMP-binding and ¿degrading 'trigger PDE' and transcription factor with a LuxR-EAL domain architecture- Structure and function of a putative c-di-GMP riboswitch in the intergenic region of the mRNA of the orf0317-pdeL operon, which down-regulates PdeL expression.Furthermore, the integration of these molecular processes within the large regulatory network that controls matrix production and thereby generates functionality and architecture of a biofilm will be elucidated.With its focus on sensory input in the control of DGCs and PDEs, c-di-GMP-sensing effector components and local c-di-GMP signaling, this project is expected to make a major contribution to achieving the goals of SPP 1879. Moreover, its results will have practical applications, e.g. in the development of novel anti-biofilm strategies or in synthetic biology.