趋化性变异系统Wsp在无鞭毛产酶溶杆菌中通过独特的c-di-GMP信号途径调控HSAF合成的机制研究

经典的细菌趋化性是指它们借助鞭毛运动，远离有害环境，趋向有利生境的行为。而变异的趋化性系统不再控制细菌的趋化运动，却分化出其他的生物学功能。Wsp是一种典型的趋化性变异系统，它在有鞭毛的细菌中参与生物膜形成。产酶溶杆菌OH11是一种无鞭毛的生防细菌，能外泌一种广谱高效的抗真菌物质HSAF。前期发现，一套完整的Wsp系统在OH11基因组中被保留，并且进化出通过由WspR-CdgL蛋白复合物介导的c-di-GMP信号途径来调控HSAF的合成。其中c-di-GMP是一种核酸类第二信使，而WspR和CdgL分别是c-di-GMP的合成酶和受体蛋白。本项目将阐明WspR-CdgL复合物形成的结构基础；解析WspR-CdgL互作受WspR的磷酸化和c-di-GMP浓度调节的机制；揭示该复合物调控HSAF合成的网络。研究结果有望揭示趋化性变异系统在无鞭毛生防细菌中实现功能与调控方式共进化的新机制。

Flagellated bacteria utilize a well-known chemosensory motility system enabling them to migrate towards a more favorable environment and to escape from undesirable condition. The flagellated bacteria could also design several chemosensory variant systems to regulate cell processes or behaviors other than motility. The Wsp chemosensory signaling is such a typical variant system, by which the flagellated bacteria, i.e. the pathogenic Pseudomonas aeruginosa could control the lifestyle switch from planktonic state to biofilm state to promote infection process. Lysobacter enzymogenes OH11 is a non-flagellated, Gram-negative bacterium with ability to kill crop fungal pathogens via secreting a heat-stable antifungal factor (HSAF) with new chemical structure and mode of action. Although strain OH11 is natively deficient in producing flagellar, it, however, retains a homologous Wsp system in the genome without unknown reason. To our surprise, the eariler findings of this project revealed that the Wsp system in the non-flagellated L. enzymogenes OH11 participated in a unique c-di-GMP signaling pathway comprising a WspR-CdgL binary complex to control the antibiotic HSAF biosynthesis. In this signaling pathway, WspR acted as a diguanylate cyclase responsible for c-di-GMP synthesis that is a ubiquitous dinucleotide second messenger, while CdgL is a c-di-GMP-binding protein required for HSAF production. Based on these discoveries, we aim, with great efforts, to illustrate the structural basis of the WspR-CdgL binding to identify key domains and/or residues of each component required for the binding with its partner. The importance of these identified key domains and/or residues in the HSAF biosynthesis and its operon expression will be investigated accordingly. Moreover, we also aim to explore whether the phosphorylation of WspR would influence its enzymatic activity to lower or promote c-di-GMP synthesis in vitro and in vivo. The effect of the phosphorylation state of WspR and its-derived c-di-GMP amounts on the WspR-CdgL binding and HSAF biosynthesis will be investigated too. Our results will uncover the mechanism by which the Wsp system evolves to regulate the antibiotic HSAF synthesis and thus provide a basis to reveal a function-mechanism co-evolved strategy of this system from flagellated to non-flagellated bacteria. Moreover, molecular modification of the Wsp signaling system to relieve the inhibition of c-di-GMP on HSAF synthesis could facilitate us to enhance HSAF yield, which further benefits the application of HSAF in biocontrol of crop fungal diseases.