联合固氮施氏假单胞菌碳氮代谢基因网络调控的分子机制

The process responsible for reduction of molecular nitrogen into ammonia via nitrogenase is referred to as biological nitrogen fixation, which is highly energy-demanding and exclusively found in prokaryotes. Thus, gene expression of nitrogen fixation ought to be tightly regulated by a complex regulatory network controlling a variety of cellular processes. Availability of both carbon and nitrogen sources are the two key regulatory signals controlling the nitrogen fixation process through the global nitrogen and carbon metabolism regulatory systems. Pseudomonas stutzeri strain A1501 that isolated from rice root has drawn great attentions, particularly due to its ability to fix nitrogen and promote rice growth by association with rice roots. This strain is one of the rare examples of nitrogen fixation within the Pseudomonas genus sensu stricto. The complete nucleotide sequence of the genome was determined and led to the identification of a 49-kb genomic island carrying the nitrogen fixation (nif) gene cluster. The genetic and physiological responses to fixed nitrogen have also been well studied in A1501 and other diazotrophic strains. Furthermore, we have preliminarily identified some non-coding RNAs (ncRNAs) that may be involved in such regulatory processes. However, the role of ncRNAs in the associative nitrogen fixation have not yet been examined using genomics analysis and/or with a combination of genetic and transcriptomic analysis. In particular, a possible molecular mechanism of regulatory network coupling between nitrogen and carbon metabolism yet to be discovered. Based on comparative genomic analysis, transcriptomic analysis under the fixed nitrogen conditions, and characterizing various genes involved in regulating carbon and nitrogen metabolisms, we propose to carry out (1) further identifying small RNAs (ncRNAs) induced under nitrogen fixation conditions and investigating their potential roles in gene network regulation; (2) understanding carbon and nitrogen signal transduction and regulatory mechanisms; (3) investigating regulatory network coupling between carbon and nitrogen metabolism for associative nitrogen fixation genes expression. Our researches on associative nitrogen fixation at molecular levels may elucidate important mechanisms on how to enhance associative nitrogen fixation activity, and therefore to pave the way to overcome fixed nitrogen suppression and insufficient carbon supply for associative nitrogen fixation under field conditions. Ultimately, it will provide a framework for future investigation on the molecular basis of complex regulatory network of nitrogen fixation, and may help to improve sustainable agriculture by reducing chemical fertilizer requirements for crops to certain extend.

联合固氮菌在长期进化中形成了一套复杂而精细的基因表达调控网络，以适应复杂而多变的外界环境。碳源和氮源种类及其浓度变化是影响联合固氮基因表达的两大环境限制因子。目前，联合固氮菌碳氮信号传导以及碳氮偶联调控的分子机制尚不十分清楚，特别是小RNA是否参与固氮基因表达的调控，还没有直接的科学证据，相关研究属于当前联合固氮研究领域的前沿和热点问题。本申请针对上述科学问题，以分离自我国南方稻田根际土壤的固氮施氏假单胞菌为研究对象，在完成其比较基因组，固氮条件下转录组以及一系列参与碳代谢和氮代谢调节相关基因功能鉴定的基础上，拟开展的研究内容包括：（1）在固氮条件下表达的小RNA的功能鉴定及调节作用研究；（2）碳信号或氮信号传导与调节机制研究；（3）碳氮代谢偶联调控固氮基因表达的分子机制研究。上述研究结果将为联合固氮基因表达网络调控机理的揭示提供重要的理论依据。

根际联合固氮菌在长期进化中形成了一套响应外界环境信号的复杂调控网络。在根际环境中，碳源和氮源变化是关键的外部信号，强烈影响联合固氮菌的固氮、逆境反应、生物膜形成等各种代谢活动。但是，目前有关联合固氮菌碳氮信号传导及碳氮偶联调控的分子机制尚不清楚。本项目以固氮施氏假单胞菌为研究对象，系统开展“固氮相关非编码RNA作用机制、碳氮代谢与信号传导机制、碳氮偶联调控机制及固氮与相关抗逆模块人工设计”等4个方面的研究工作，取得如下重要进展：（1）发现了53个在固氮条件下可检测的特异感应外界氧、氮及碳信号的非编码RNA，包括两个全新的直接参与固氮调控的非编码RNA：nfiS和nfiR。在进化过程中，固氮酶基因通过“招募小劳动模范”非编码RNA如nfiS和nfiR，增强固氮酶基因mRNA稳定性和转录效率，进而实现最佳固氮活性；（2）提出了碳氮调控系统Rpo/Gac/Rsm介导的生物膜形成新机制，证明RpoN在固氮施氏假单胞菌生物膜形成“抱团耐铵固氮效应”中的核心调控作用。在低氮/碳丰富条件下，氮调控因子RpoN通过正调控pslA和nifA等基因的转录，而感应碳源丰富信号的非编码RNA如RsmZY通过与RsmA结合，解除RsmA对algU和sadC基因表达的转录后抑制，促进生物膜的形成；（3）证明混合碳源条件下A1501具有典型的“挑食有机酸效应”, 即优先利用有机酸如琥铂酸等优势碳源，同时其它非优势碳源如葡萄糖代谢途径基因表达则被抑制。在优势碳源存在时，碳代谢物抑制蛋白Crc与非优先碳源代谢途径基因的mRNA结合抑制其转录后表达，一旦优势碳源消耗殆尽，非编码RNA如CrcZY与Crc结合，使其从靶标mRNA脱离并解除其抑制作用，细胞开始利用非优势碳源；（4）从特殊微生物基因资源中克隆了一系列具有固氮岛、铵转运、分子伴侣等特性的新基因（簇），成功实现在大肠杆菌和固氮微生物中的异源表达，增强其抗逆和固氮的能力。上述研究为揭示联合固氮基因表达网络调控机理奠定了重要的工作基础，对于大幅度提高田间联合固氮效率具有理论指导意义。