恶臭假单胞菌KT2440中c-di-GMP合成酶WspR上游转录调节子的筛选和功能探究

环二鸟苷酸(c-di-GMP)是细菌中普遍存在的核苷类第二信使，参与调控生物被膜形成等生理活动。WspR是假单胞菌中保守的c-di-GMP合成酶，在c-di-GMP介导的对生物被膜的调控中起关键作用。WspR的功能和作用机制已有广泛研究，但对于其转录调控知之甚少。申请人前期揭示了恶臭假单胞菌KT2440中WspR调控生物被膜形成的机制，且发现缺失sigma因子RpoS导致wspR转录显著下降。在此基础上，本项目将利用5’RACE和体外转录等手段深入探究RpoS在wspR转录调控中的作用和机制；同时运用细菌单杂交和凝胶迁移阻滞技术在基因组范围内筛选WspR上游的转录调节子，并解析其对wspR表达、胞内c-di-GMP水平及生物被膜形成的影响和作用机制。研究结果将为揭示假单胞菌调控c-di-GMP水平及生物被膜形成的机理奠定基础，并拓展对以c-di-GMP为核心的调控通路的全面认识。

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial secondary messenger that involved in a diverse range of cellular processes including biofilm formation. Synthesize of c-di-GMP is done by diguanylate cyclase (DGC) enzymes. WspR is a conserved DGC in Pseudomonas species, which plays a vital role in regulating biofilm formation. Function mechanism of WspR has been widely studied, but transcriptional regulation of wspR remains poorly understood. In previous studies, we discovered the mechanism by which WspR regulating biofilm formation in Pseudomonas putida KT2440, and we also found that transcription of wspR showed an obvious decrease in RpoS mutant. In this study, role of RpoS in transcription of wspR will be further confirmed by 5’RACE and in vitro transcription assay. Meanwhile, this work attempts to screen and characterize the regulators that control wspR transcription using bacteria one-hybrid system and electrophoretic mobility shift assay. Further research about the influence of target regulators on expression of wspR, intracellular c-di-GMP levels and biofilm formation will also be performed. The results of this study will be useful in elucidating the regulation mechanisms of c-di-GMP level and biofilm formation in P. putida, meanwhile, it will deeper our understanding on regulatory pathways based on c-di-GMP.