氮代谢调控因子GlnR通过cda操纵子调控细胞渗透平衡的新机制

渗透平衡是细菌细胞正常生长所必需的。在厚壁菌门中，cda操纵子结构保守，编码c-di-AMP合成酶CdaA、调控蛋白CdaR及肽聚糖合成的关键酶——磷酸葡萄糖胺变位酶GlmM。前期研究表明，在蜡样芽胞杆菌群中，cda操纵子的启动子区域存在氮代谢调控因子GlnR的结合基序，并能与之结合。本研究拟以苏云金芽胞杆菌BMB171为材料，通过构建glnR缺失株、过表达株及操纵子缺失株，揭示GlnR通过谷氨酰胺合成酶GS介导响应不同氮浓度调控cda操纵子的表达，从而“一箭双雕”调节胞内CdaA和GlmM的丰度，分别影响c-di-AMP与肽聚糖合成中间代谢物的浓度，进而调节钾离子运输和肽聚糖合成，最终“合二为一”协同调控细胞渗透平衡的作用机制。本项目不仅揭示GlnR响应不同氮浓度调控cda操纵子的新机制，阐明氮对细胞渗透平衡的影响，丰富c-di-AMP的调控网络，也为蜡样芽胞杆菌群的应用研究提供新思路。

Osmotic balance is necessary for the normal growth of bacterial cells. In the Phylum Firmicutes, the cda operon (cdaAcdaRglmM operon) structure is highly conserved and encodes c-di-AMP synthetase CdaA, the regulatory protein CdaR, and the key enzyme for the peptidoglycan synthesis (phosphoglucosamine mutase GlmM). Previous studies have shown that in the Bacillus cereus group, there exists a binding motif for GlnR (the transcriptional regulator of nitrogen metabolism) in the promoter region of the cda operon and that they can bind closely. In fact, GlnR is widely present in Gram-positive bacteria, and can sense extracellular glutamine mediated through glutamine synthetase (GS) to inhibit the transcription of downstream genes, thereby regulating the absorption and utilization of nitrogen sources. We thus intend to study how GlnR regulates the cda operon via responding to different nitrogen concentrations of Bacillus thuringiensis BMB171 by constructing a series of glnR deletion strain ΔglnR, overexpression strain OEglnR, and cda operon deletion strain ΔcdaAΔcdaRΔglmM, and by using RT-qPCR, β-galactosidase activity assay and Western blotting methods, etc. to reveal how inhibiting the expression of CdaA can result in reduced c-di-AMP synthesis to affect the cellular osmotic pressure on the one hand; also, we want to know how inhibiting the expression of GlmM can affect the synthesis of peptidoglycan on the other hand, and ultimately leads to synergistic regulation of the cellular osmotic balance. This project will thus not only reveal a new mechanism for understanding how GlnR responds to different nitrogen concentrations to regulate cda operon but also how varying nitrogen source can regulate cellular osmotic balance, enrich the regulation network of c-di-AMP, and provide a new idea for the application research of Bacillus cereus group.