双组份系统是细菌的主要信号传导系统，可调控细菌的大多数生理过程。初步研究表明，天蓝色链霉菌SCO2120/2121系统的突变可导致抗生素产量和孢子颜色的变化，说明SCO2120/2121可能是一个新的全局性调控因子。本项目以此为基础，开展SCO2120/2121系统调控天蓝色链霉菌生理过程的机制的研究。首先，敲除SCO2120/2121，确定该系统的生物学功能；然后，提取野生菌和突变菌的总RNA，进行全转录组测序，界定该系统所调控的基因集群；构建带标签的SCO2120表达质粒，通过染色质免疫共沉淀，富集SCO2120结合的DNA小片段，进行高通量测序，定位SCO2120在染色体上的所有结合位点；结合转录组和免疫沉淀测序数据，选取合适靶基因，利用凝胶迁移和足迹印记技术，分析SCO2120的保守结合序列；最后，通过磷酸基团的转移实验，探索SCO2120/2121与其它双组份系统之间的交流。

Two-component signal transduction systems (TCS) are responsible for sensing and responding to the environment of cells and are central to much of the cellular physiology that results from alterations in the enviroment. Streptomyces produce an impressive amount of secondary metabolites, many of them are antibiotics in clinical use. Our preliminary research showed that mutation of SCO2120/2121 TCS leads to decreased production of the antibiotics actinorhodin (ACT) and undecylprodigiosin (RED) and delayed sporulation in Streptomyces coelicolor, suggesting that SCO2120/2121 is a new global regulator of Streptomyces coelicolor. Based on the preliminary data, this project will focus on defining the regulatory role of SCO2120/2121. We will first determine the role of SCO2120/2121 TCS by knocking out their encoding genes and comparing the phenotype of the wild-type and mutant strains. Secondly, the regulon of SCO2120/2121 TCS will be investigated by whole transcriptome sequencing of the wild-type and mutant strains and by comparing the transcriptome data of these two strains. Genes belonging to the SCO2120/2121 regulon, as determined by significant differences between the mutant and wild-type strains, will be categorized using bioinformatics to deduce the potential physiological roles of SCO2120/2121. The in vivo binding site of the response regulator SCO2120 on the chromosome of Streptomyces coelicolor will be mapped by Chip-seq analysis. To achieve this goal, an inducible and tagged SCO2120 expressing plasmid will be constructed and introduced into the mutant strain, thereby creating an engineered strain for Chip-seq assay. The engineered strain will be induced and processed to map all of the binding locations of SCO2120 on the chromosome of Streptomyces coelicolor. Genes of interest that are differentially expressed in the mutant by tanscriptome sequencing analysis, and whose promoter is also occupied by SCO2120 in the Chip-seq assay will be selected for further analyses. EMSA analysis and DNase I foot-printing assays will be performed to localize the promoter region protected by SCO2120, and the consensus binding site for SCO2120 will be deduced. Finally, cross-talk between SCO2120/2121 and other TCSs of Streptomyces coelicolor will be investigated by phosphorylation assays, to determine if there is cooperation among the TCSs. This research will provide new insights into the regulation of antibiotic synthesis and other physiological processes in this medically important genus.