分枝杆菌氮代谢信号转导途径中关键基因的筛选及其功能鉴定

In actinomycetes, nitrogen metabolism is a matter of great concern because it is not only closely related to pathognesis in Mycobacterium tuberculosis but also greatly influences the yield of secondary metabolites in many important industrial actinomycetes, e.g. Streptomyces. Distinct from Escherichia coli, the nitrogen metabolism in actinomycetes is governed by GlnR, an orphan response regulator. Up to date, most studies in nitrogen metabolism are being conducted on the characterization of GlnR target genes, GlnR binding cis-elements and the post-translational modification of GlnR, while little is known about the signal receptor and the signal transduction pathway involved in nitrogen limitation stress. To answer these scientific questions, this project is designed to conduct a systematic study on the nigrogen metabolism in Mycobacterium smegmatis, a model actinomycete strain for studying Mycobacterium. A highly efficient transposon mutation method will be employed, and different nitrogen culture conditions will be used to screen mutants that fail to survive under nitrogen-limited conditions. Moreover, the next generation sequencing strategy will also be taken to find candidate genes, which will be further characterized about their physiological roles in nitrogen signal recognition and transduction. In summary, we hope to construct a global regulatory network of nitrogen metabolism in actinomycetes, which may further facilitate the investigation of the relationship between nitrogen metabolism and pathogenesis in pathogenic actinomycetes, e.g. M. tuberculosis.

在结核分枝杆菌中，氮代谢因与致病性紧密相关而备受关注；在其它的重要工业放线菌中，氮代谢也影响抗生素的产量，其重要性不言而喻。与经典的大肠杆菌氮代谢调控系统不同，放线菌的氮代谢调控由全局性氮代谢调控蛋白GlnR负责。目前对于放线菌氮代谢的研究基本上集中于鉴定GlnR的靶标基因及GlnR结合靶标DNA的顺式元件；对GlnR蛋白的翻译后修饰也有一部分研究；但是，对于放线菌氮匮乏信号的感受以及相关的信号转导途径都少有研究。本项目将在前期工作的基础上，以模式放线菌耻垢分枝杆菌为主要研究对象，利用已经建成的高效转座子插入失活突变库，并结合高通量测序的方法来筛选参与氮代谢信号识别和信号转导的关键基因。在此基础上，利用分子生物学、生物化学等手段对筛选到的新基因进行进一步的功能鉴定，进而最终解析放线菌全局性氮代谢调控网络，为研究分枝杆菌氮代谢与致病性的关系提供重要的理论指导。

在以结核分枝杆菌为代表的致病性放线菌中，其致病性与氮代谢紧密相关。现有研究表明，放线菌的氮代谢调控与经典的肠杆菌不同，其主要受到全局性氮代谢调控因子GlnR的转录调控。然而，目前还不清楚，在氮源匮乏的条件下，分枝杆菌是如何感受氮源信号并做出应答反应的？为此，本项目中，我们开发了基于转座子技术和高通量测序技术的功能基因组方法，利用不同氮源条件下，转座子插入突变库的差异来筛选与贫瘠氮源代谢相关的未知基因。通过生物信息学比对，基因敲除，回补，以及一系列的体外实验，我们证明了一对双组份系统，pdtaRS，参与了氮源匮乏信号的转导。通过对双组份系统下游基因的鉴定，我们发现并证明了PdtaRS通过调控gltBD的转录，进而参与了谷氨酸代谢的调控，而其深入的调控机制还在进一步研究之中。总结来说，本项目通过功能基因组学技术鉴定了一条很可能位于全局性氮代谢调控蛋白GlnR上游的PtdaRS通量，有助于进一步解析整个氮代谢调控通路。此外，为了便于放线菌的基因功能研究，我们还开发了一套基于CRISPR-Cas12a技术的基因编辑系统，从而为后续的研究打下了坚实的基础。本项目发表SCI论文4篇，培养了7名硕博士研究生。