DNA复制起始调控对于结核分枝杆菌的潜伏感染和在宿主中的生存十分关键，然而相关分子机制尚不清楚。申请人团队前期研究发现结核分枝杆菌双组份系统激酶TrcS与复制起始蛋白DnaA之间存在相互作用，并且二者之间存在磷酸转移现象。本项目将结合遗传学和分子生物学技术手段系统研究TrcS磷酸化修饰DnaA的分子机制及其对结核分枝杆菌应对环境压力的作用机制。首先采用质谱分析技术鉴定DnaA的磷酸化位点；进而应用细菌双杂交系统、Co-IP及SPR技术鉴定TrcS磷酸化修饰DnaA的分子机制；最后通过监测环境胁迫条件下trcS基因异常表达菌株的生长变化情况及菌株中DnaA的磷酸化状态，鉴定TrcS磷酸化修饰DnaA的生理功能。研究结果将阐明结核分枝杆菌的DnaA磷酸化修饰调控机制及对病原菌应对环境胁迫的作用机制。

DNA replication initiation regulation is essential for dormant infection and survival of Mycobacterium tuberculosis in the host. However, our understanding on underlying molecular mechanism is still largely incomplete. In previous studies, we found an interaction between kinase TrcS of two component system and replication initiator DnaA in Mycobactium tuberculosis. In addition, the phenomenon of phosphate transition was embraced by TrcS and DnaA. This project will focus on systematically studying the molecular mechanism of phosphate transition from TrcS to DnaA, and its effective mechanism of Mycobacterium tuberculosis responding environmental stresses. First, phosphorylation sites of DnaA will be identified by mass spectrometry. Second, molecular mechanism of phosphate transition from TrcS to DnaA will be illustrated by bacterial two-hybrid system, Co-IP and SPR assays. Third, the physiological roles of phosphate transition from TrcS to DnaA will be analyzed by identifying the bacterial growth and morphology of strains expressing abnormally TrcS under stressful conditions and phosphorylation of DnaA with these strains. The results will elucidate the regulation mechanism of phosphorylation of DnaA in Mycobacterium tuberculosis and its roles on pathogen responding environmental stresses.