地衣芽胞杆菌群体感应系统ComQXPA调控铁摄取机制的研究

Quorum sensing (QS) is an important regulatory system for Bacillus licheniformis to sense environmental changes and coordinate cellular responses. Previous studies of applicants have found that ComQXPA, a quorum sensing system of Bacillus licheniformis, can respond to iron deficiency stress and has extensive regulatory effects on genes involved in iron uptake pathway. Investigating the mechanisms of ComQXPA in response to iron deficiency stress and the regulation of ComA for iron uptake genes is of great significances for revealing iron homeostasis in Bacillus species. It will be helpful to elucidate the role of quorum sensing system in environmental adaptation of Bacillus species. Based on the native strain B. licheniformis DW2 and its derivative strains, this project will excavate and analyze the below mechanisms: the effects of iron concentration on promoter efficiency of comQX、comP、comA genes, the catalytic activity of ComQ and the DNA binding activity of ComA，by the methods of electrophoretic mobility shift assay (EMSA)，promoter replacement and enzyme activity assay etc. Then the regulation of ComA and DegU on iron transporter genes, siderophore synthesis and transporter gene will also be analyzed by the methods of EMSA, DNase I footprinting, bacteria single-hybrid assay, and GFP expression assay. Finally, this project will clarify the mechanism of quorum sensing system responding to iron deficiency stress and regulating iron uptake in Bacillus species.

群体感应是地衣芽胞杆菌感知环境变化，协调细胞应答反应的重要调控系统。申请人前期研究发现地衣芽胞杆菌群体感应系统ComQXPA能够独立响应铁离子浓度的诱导且对铁摄取途径基因具有广泛的调控作用。解析ComQXPA应答缺铁胁迫压力及调控细菌铁元素摄取机制，对阐明群体感应系统在芽胞杆菌适应缺铁环境中的作用具有重要意义。本项目以地衣芽胞杆菌DW2为出发菌株，首先通过启动子替换、体外催化实验、凝胶阻滞实验等手段解析铁离子浓度对comQXPA基因簇各个启动子效率、ComQ催化活性、ComA蛋白DNA结合能力的影响；通过凝胶阻滞、DNA酶I足迹、细菌单杂交、报告基因表达等方法解析转录因子ComA及其下游转录因子DegU对铁离子通道蛋白基因、铁载体合成及转运基因的调控机制。上述成果将为阐明群体感应系统应答缺铁胁迫并调控芽胞杆菌铁摄取行为机制及解析芽胞杆菌对缺铁胁迫环境的适应过程奠定基础。

群体感应是地衣芽胞杆菌感知环境变化，协调细胞应答反应的重要调控系统。其通过直接调控或者调控下游调控因子的表达间接调控细胞的多条代谢途径。本项目证实了铁离子浓度是诱导ComQXPA基因簇启动子的重要环境因子。进一步通过基因敲除、过表达、GFP报告蛋白表达分析、RT-qPCR、EMSA等实验挖掘了受ComA和DegU调控的铁摄取相关的靶基因。鉴定了ComA和DegU对铁摄取相关基因的调控模式，其中包括负调控dhbA、feuA、besA、yhfQ、fhuB、ykuN、yfiZ、yvmC、yvnA并正调控dps、yfmD、yfiy、yvmB等。阐明了ComA和DegU在不同铁离子浓度环境下调控铁载体bacillibactin和铁离子螯合剂普切明酸合成及转运的机制。在缺铁条件下，地衣芽胞杆菌可通过调降DegU的表达水平来提高bacillibactin的合成量，并抑制普切明酸的合成，增强Fe3+-铁载体复合物以及游离铁离子向胞内的转运，释放白内储存的铁离子，从而满足菌株生长和代谢等生命活动的供铁需求。该研究成果将为进一步解析地衣芽胞杆菌应答缺铁胁迫并调控铁摄取行为机制及解析芽胞杆菌对缺铁胁迫环境的适应过程具有重要意义。