施氏假单胞菌非编码RNA nfiS参与固氮基因表达调控的分子机制

To respond to multiple environmental cues, nitrogen-fixing bacteria have evolved sophisticated regulatory networks to adjust expression of nitrogen fixation genes at the transcriptional level. The ammonium ion, oxygen concentration and energy supply are the most three important environmental signal that affect the nif gene expression. Small non-coding RNAs (ncRNAs) that play important regulatory roles exist in numerous organisms but few were functionally characterized in nitrogen-fixing bacteria. Supported by the former NSFC, we identified 17 ncRNAs that were upregulated under nitrogen fixation conditions compared with nitrogen excess conditions in the root-associated nitrogen-fixing bacterium Pseudomonas stutzeri A1501 using a deep sequencing approach. Among those ncRNAs, nfiS can specifically respond to oxygen signals and its expression was under the control of RpoN, a well-studied nitrogen regulator. Mutation of nfiS not only impaires the nitrogenase activity but also decreases the expression of the nif genes, implies that nfiS might be involved in the nitrogen fixation regulation process. In order to investigate the regulatory mechanism of nfiS involved in the nitrogen fixation gene regulation and the mode of its response to external signals, the nfiS disruption mutant and overexpression strain will be constructed in this study; qRT-PCR and EMSA will be performed to research the expression characteristics of nfiS and its location in the regulatory system of nitrogen fixation; the proteome will also be employed to analyze and verify the target genes of nfiS. Ultimately, the roles of nfiS in the nitrogen fixation regulation will be determined. This work will lay an important theoretical foundation for the in-depth analysis of the expression regulation mechanism of nif genes.

固氮基因的表达调控严谨而高效，受到外界氧、氮及能量等多个因素的影响。具有调控功能的非编码RNA在细菌中被大量发现，广泛参与了物质代谢、胁迫反应等重要生理过程，但在固氮微生物中鲜有报道。在上一个基金项目的支持下，我们通过转录组分析发现施氏假单胞菌A1501在固氮条件下高表达的17个非编码RNA，其中非编码RNA nfiS可特异响应外界的氧信号，而且其表达依赖于固氮代谢调控sigma因子RpoN，nfiS的突变降低了固氮酶活性和固氮结构基因的表达水平，暗示该基因可能以某种机制参与了固氮调控。本研究拟通过：1）功能互补、启动子活性测定等方法明确nfiS的功能特征及在固氮调控网络中的定位；2）采用转录组、蛋白质组技术研究nfiS的调控网络并寻找其作用靶标；3）通过qRT-PCR、凝胶阻滞技术研究nfiS与靶标基因互作的分子机制。研究结果将为深入解析固氮基因表达调控机理奠定重要的理论基础。

非编码RNA广泛参与了胁迫抗性、毒力、运动、生物膜形成、群体感应和代谢调控等过程，在转录后水平发挥调控作用，但是直接参与固氮调控的非编码RNA未见报道。本课题在固氮施氏假单胞菌A1501中鉴定了一个新的具有种特异性的非编码RNA NfiS, 通过突变株构建及表型分析明确了NfiS的功能，进一步的RT-PCR及启动子-蛋白互作研究了NfiS的转录及表达特征，通过蛋白质组分析及生物信息学预测确定了NfiS在固氮调控中的作用靶标并采用生物大分子互作技术验证了其相互作用，探讨了NfiS参与固氮基因调控的分子机制。取得以下主要结果：.（1）Northern杂交证实nfiS基因是典型的非编码RNA，在固氮条件下高表达，nfiS突变造成菌体固氮酶活及氧化胁迫能力显著降低，相关基因表达下调；（2）5'-RACE实验确定了nfiS的转录起始位点和转录方向，nfiS的启动子为RpoN依赖型；（3）蛋白组学分析及质谱鉴定确定了NfiS参与固氮调控的靶标为固氮酶编码基因nifK的mRNA；二级结构分析发现NfiS的茎环上存在与nifK mRNA核苷酸片段互补配对的位点，而且此茎环结构是固氮施氏假单胞菌所特有；（4）非固氮施氏假单胞菌ATCC17588菌株的nfiS同源序列不能回补A1501 nfiS突变株的固氮表型，但能够回补氧化胁迫抗性表型，表明11bp特有区段对于nfiS调控固氮的表型是必须的；（5）采用微量热涌动确定了NfiS与靶标nifK mRNA存在体外直接互作，且发现NfiS的突变造成靶标nifK mRNA的半衰期显著缩短。综合以上研究结果，绘制了A1501菌中NfiS参与固氮基因表达调控的作用模型：NfiS可特异响应外界固氮和渗透胁迫信号，受氮代谢调控sigma因子RpoN的调控，通过RNA-RNA互作特异的与nifK mRNA结合，保护nifK mRNA的稳定性，从而提高nifK mRNA的翻译效率，实现对固氮酶活性水平的调控。以第一作者或通讯作者发表学术论文12篇，其中SCI收录5篇，中文核心期刊7篇，非编码RNA nfiS调控固氮基因表达的工作获得重要理论突破，论文发表在美国科学院院刊（PNAS）。本研究成果将有助于提高联合固氮效率、打破环境因素抑制，为开发高效固氮的基因工程菌株提供理论支撑。