组氨酸激酶NisK感应nisin及进行信号传导的分子机制

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.

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。

组氨酸激酶NisK可感应胞外的羊毛硫细菌素乳链菌肽（nisin）来调控nisin本身的生物合成。Nisin由34个氨基酸组成，分子中含有五个硫醚环。NisK蛋白含有两个跨膜域和一个较大的胞外区，代表了一类特殊的组氨酸激酶。他们的结构与大多数能感应肽类信号的多跨膜域组氨酸激酶不同，他们接受肽类信号的分子机制更是很少有报道。为了揭示NisK蛋白如何感应nisin分子，我们构建了一株乳酸乳球菌报告菌株。在此菌株中，双组份调控系统nisRK组成型表达，报告基因lacZ受nisA基因的启动子控制，可被nisin诱导进行表达。利用此菌株进行far-western blot实验，结果证明NisK和nisin是可以相互作用的。同时，缺失突变分析证明NisK的胞外区是识别感应nisin的关键区域。经过比对，我们发现NisK的胞外区有一些保守位点，在报告菌株中将这些保守位点进行丙氨酸替换突变，用nisin诱导突变体菌株，检测报告基因的表达水平，结果发现有几个疏水性的保守位点对感应nisin是非常重要的。接着对NisK胞外区的几个疏水区也进行替换突变，发现胞外区的第一个β折叠是NisK识别nisin的很重要的疏水区域。将重要氨基酸分别替换成不同性质的氨基酸后进行诱导实验，证明疏水作用确实是NisK识别nisin的主要作用力。同时我们还建立了半体外合成系统，可在大肠杆菌中方便的合成诱导分子nisin的突变体，并利用此方法合成了大量nisin的丙氨酸扫描突变体。同样利用构建报告菌株、检测报告基因表达来验证突变体的诱导活性，从而鉴定nisin识别NisK的关键结构或位点。结果表明，nisin的A环和B环是识别NisK的主要结构。由此我们提出了含两个跨膜域的组氨酸激酶NisK通过疏水作用感应识别羊毛硫细菌素nisin的分子模型。我们的结果填补了双跨膜域组氨酸激酶感应肽类信号分子机制的空白，扩大了我们对组氨酸激酶如何感应外界信号并调控细胞生理反应的理解。