乳链菌肽nisin是羊毛硫细菌素典型代表，已作为安全生物防腐剂被广泛应用。前期研究表明nisin通过双组份信号系统NisK/R进行自调控合成，但其信号传导的开启即组氨酸激酶NisK对信号分子nisin感应及信号传导的分子机制尚不清楚。NisK蛋白N端感应区无保守感应结构域，暗示可能存在特殊的信号感应机制，而其第二个跨膜域两端含有极性氨基酸簇，推测可能影响信号从胞外到胞内的传导。本申请拟主要围绕NisK对nisin的感应机制，一方面通过晶体解析及构建NisK突变体报告菌株等体内体外手段研究NisK胞外感应区结构及识别nisin的位点，另一方面通过无细胞体外翻译合成nisin突变体深入研究nisin分子中与NisK结合的位点。同时通过对NisK跨膜域氨基酸进行突变筛选研究信号从NisK胞外到胞内传导的机制，为全面理解羊毛硫细菌素生物合成规律、进而为理解微生物对外界信号的识别感应及传导奠定基础。

Nisin is a prototype lantibiotic, and has been widely used as a safe preservative. Biosynthesis of nisin was reported autoregulated by two-component signal tranduction system NisK/R, however, the nechanism of histidine kinase NisK sensing and transducting the signal nisin is unknown. Histidine kinases usually recoganize and sense periplasmic signals by the specific N-terminal periplasmic sensor motifs,however,no conservative sensor motif is found in NisK N-terminal region, indicating that NisK may sense the signal nisin with new mechanism. Two polar amino acid clusters in the second transmembrane region in NisK are supposed to be invovled in periplasmic to cytoplasmic signal transduction. This application focus mainly on the research of NisK sensing mechanism.The structure of NisK N-terminal periplasmic region is supposed to be clarified using protein crystal structure analysis or protein affinity technology in vitro to find a new sensor motif of NisK. The following construction of NisK mutants reporter strains to check the downstream reporter gene expression in vivo would make sure the function of the new sensor motif.Nisin mutants would be synthesized using rapid translation system in a short time easily,and be used to interact with NisK to find its binding sites or regions.In this application NisK periplasmic to cytoplasmic signal transduction mechanism will also be discussed using amino acids replacement screening in the second transmembrane region of NisK. The research of NisK sensing and transducting the signal nisin in this application will help to understand more about lantibiotics synthesis and regulation, and the mechanism of bacterial recognizing,sensing and transducting periplasmic signals will be well known.