恶臭假单胞菌中GcbA上游激酶配体的筛选及功能鉴定

GcbA is a conserved c-di-GMP synthase that consists of two N-terminal REC domains and a C-terminal GGDEF domain in Pseudomonas species, participating in regulation of initial-biofilm formation and flagella-mediated swimming. Phosphorylation of the REC domain significantly promotes the synthesis ability of GcbA. However, the kinase partner responsible for GcbA phosphorylation has not been identified. To seek kinase ligands of GcbA, we constructed a protein expression library including all kinases in Pseudomonas putida KT2440, and then GcbA-interacting kinases were screened by using bacteria two-hybrid system. Eight potential target kinases were obtained in the screening. On this basis, interaction of GcbA with the eight kinases will be further confirmed by immunoprecipitation and in vitro protein-protein binding assay. Meanwhile, this work also attempts to investigate the binding properties of kinase to GcbA by isothermal titration calorimetry and in vitro trans-phosphorylation assay. In addition, effects of these kinases on enzymatic activity of GcbA and strain phenotypes (biofilm formation and swimming motility) will also be analyzed. The results of this study will improve our understanding on function of GcbA in Pseudomonas putida KT2440, and will be useful in elucidating the regulatory pathways based on GcbA.

GcbA是假单胞中保守的c-di-GMP合成酶，由N端两个REC结构域和C端GGDEF结构域构成，调控初期生物被膜形成和鞭毛介导的游动性。REC结构域的磷酸化导致GcbA合成c-di-GMP的能力显著增强，但负责GcbA磷酸化的激酶至今未被发现。为寻找GcbA上游激酶配体，申请人前期构建了恶臭假单胞菌KT2440中所有激酶的蛋白表达库，并通过细菌双杂交筛选得到8个与GcbA互作的激酶。在此基础上，本项目将继续通过免疫共沉淀和蛋白质体外结合等技术验证前期得到的激酶与GcbA的互作，并利用等温滴定量热法和体外磷酸转移探究激酶与GcbA的结合特性；同时，将通过体外酶活检测和表型观察等手段探究激酶对GcbA的代谢酶活性及下游生物被膜与运动性表型的影响，揭示激酶配体的生理功能和作用机制。研究结果将有助于完善对KT2440中GcbA功能的认识，并为全面揭示以GcbA为核心的信号响应通路奠定基础。

GcbA是假单胞菌中发现的保守的c-di-GMP合成酶，由N端REC结构域和C端GGDEF结构域构成。gcbA缺失及超表达后显著影响鞭毛介导的运动性，但GcbA调控运动性的机制未知。当将GcbA上“GGDEF”模序突变成“GGAAF”后，GcbA合成活性丧失。同时发现，GcbA依赖其合成活性调控运动性。进一步研究发现，GcbA的REC结构域上接收磷酸基团的天冬氨酸位点D86和D212同时突变后显著降低KT2440的运动性。为寻找能将GcbA磷酸化的上游激酶配体，本研究构建了KT2440中67个激酶的蛋白表达库，并通过细菌双杂交筛选到了10个与GcbA互作的激酶蛋白。GST-Pull-down实验进一步确定了7个与GcbA互作的蛋白，分别是PP_1492、PP_2354、PP_3651、PP_3758、PP_3835、PP_3968和PP_4338（CheA）。接着，通过体外磷酸转移实验找到了可磷酸化GcbA的上游激酶配体CheA，并且发现CheA磷酸化GcbA后显著抑制GcbA的蛋白酶活性。CheA为趋化性信号转导系统中调控趋化性运动的核心蛋白，其编码基因缺失后显著抑制细菌的运动性。为确定GcbA与CheA在调控运动性上的关系，实验检测了cheA与gcbA单缺失与双缺失突变体的运动性并比较了它们的运动性表型差异。研究结果发现，cheA与gcbA双缺失突变体的运动性表型与cheA单缺失突变体的运动性表型一致，而与gcbA单缺失突变体的运动性表型相反，说明GcbA依赖CheA调控KT2440的运动性。此外，另外6个不能磷酸化GcbA的激酶蛋白与GcbA互作促进GcbA的蛋白酶活性，同时发现激酶PP_3835与GcbA互作抑制CheA与GcbA之间的磷酸转移。本文的研究结果确定了磷酸化GcbA的上游激酶配体CheA，并揭示了GcbA调控运动性的机制。研究结果拓展了我们对GcbA及CheA功能的认识，并为进一步全面揭示以GcbA-CheA为核心的信号响应通路奠定基础。

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