天蓝色链霉菌的一个新的具有全局调控作用的双组份信号传导系统SCO2120/2121的调控机制研究

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.

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。

细菌的双组份信号传导系统主要负责对环境信号的感应和传导，可以调控细菌的多数生理过程。链霉菌的模式菌株-天蓝色链霉菌-的基因组编码六十七对双组份信号传导系统，其中多数系统的功能未知。本研究聚焦天蓝色链霉菌中一个未知功能的双组份系统-SCO2120/2121，对该系统的生物学功能及其调控机制进行了深入研究。基于本研究结果，我们将SCO2120/2121命名为MacRS（Morphogenesis and actinorhodin regulator/sensor）。我们的研究结果表明，不论是MacRS的单突变还是双突变都会阻断天蓝色色素ACT的产生，但同样的突变却导致气生菌丝的形成提前；在成功构建具有功能的表达MacR-FLAG融合蛋白的基因工程菌株的基础上，我们进行了免疫共沉淀测序分析（Chip-Seq），从而揭示了MacR的三个体内（in vivo）结合位点（靶基因）；通过足迹印迹分析，我们确定了MacR在三个靶基因上游的保护序列，并初步推测了MacR的保守结合（识别）序列（TGAGTACnnGTACTCA，带有两个由七个碱基组成的倒转重复序列）；通过序列分析，我们在天蓝色链霉菌的基因组上发现了另外六个与MacR保守序列完全一致或高度相似的新位点，这些位点一般位于脂蛋白或膜蛋白基因的上游；通过体外（in vitro-凝胶迁移和足迹印迹分析）和体内（Chip-qPCR）分析，我们确认这六个预测的位点也是MacR的体内结合位点（即靶基因）；转录分析表明，MacR显著调控带有上述位点的靶基因的表达；SCO4011、SCO4924和SCO6728的突变可以不同程度的促进气生菌丝的形成，说明这些MacR靶基因是新的形态膜蛋白（morphogenic membrane protein）；细菌双杂交分析表明，三个形态膜蛋白之间及膜蛋白与SCO4225（也是MacR的靶基因）之间均存在相互作用；特别重要的是，MacRS的同源基因可以互补macRS突变菌，说明MacRS的功能保守性。综上所述，我们的研究确定了MacRS的生物学功能，揭示了MacRS发挥调控作用的核心元素，增加了对双组份系统的新认识。