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

细菌生物膜定植在不同的表面，对抗生素或宿主免疫系统具有高度抗性。在这些生物膜中，细胞被嵌入在细胞外生物聚合物的自我产生的保护基质中。大肠杆菌的主要生物膜基质成分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的控制。后者由二胍酸环化酶（DGC，具有GGDEF结构域）产生，并被特定的磷酸二酯酶（PDE，具有EAL结构域）降解。大肠杆菌K-12的29个GGDEF/EAL结构域蛋白中有25个具有酶活性，其中12个为DGCs， 13个为pde。许多这些差异表达的DGCs和pde与膜相关，其活性受n端感觉结构域控制。利用生物膜基质生产作为c-di-GMP的靶标，为遗传、分子生物学和生化分析提供方便的表型和读数，本项目旨在阐明大肠杆菌c-di-GMP信号在一级和二级水平上的信号感知和处理的分子机制。这包括(i)通过新型pde的n端感觉输入域感知环境和细胞信号，以及（ii）通过新型和非正统的c-di-GMP效应机制感知c-di-GMP。特别是，以下信号感知和c-di-GMP效应机制将被研究：-通过在具有CSS-EAL结构域结构的6种大肠杆菌PDEs的质周环域中形成二硫键和蛋白水解加工对细胞质酶活性的氧化还原控制- YbjK的分子结构，功能和靶标，一种deor样转录因子，似乎与c-di-GMP结合为g -四重体- PdeL的转录调节，一种局部作用的c-di-GMP结合和降解的“触发PDE”和转录因子，具有LuxR-EAL结构域结构- orf0317-pdeL操纵子mRNA基因间区推定的c-di-GMP核糖体开关的结构和功能，其下调PdeL表达。此外，这些分子过程在控制基质生产的大型调控网络中的整合，从而产生生物膜的功能和结构，将被阐明。该项目将重点研究控制DGCs和pde的感觉输入、c-di-GMP传感效应组分和局部c-di-GMP信号传导，有望为实现SPP 1879的目标做出重大贡献。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.