施氏假单胞菌铁吸收调节蛋白Fur调控固氮基因表达的分子机制

In gram negative bacteria, the intracellular iron transport, storage and utilization are mainly regulated by the ferric uptake regulator Fur. A large number of studies have shown that Fur plays important roles in iron transport, oxidative stress, microbial host interaction. Iron is an important component of mature nitrogenase, the abilities of nitrogen fixing bacteria to uptake and efficiently utilize the iron element are crucial to the function of nitrogenase, but there is no report on whether Fur is directly involved in the regulation of nitrogen fixation. In our previous research, it was found that the nitrogenase activity of the fur mutant was sharply decreased with 90% of the wild type in the nitrogen fixing Pseudomonas stutzeri A1501. The subsequent qRT-PCR analysis showed that the expression of both the nitrogenase component gene nifH and the nif-specific regulator gene nifA were decreased, however no significant changes were found to the general nitrogen regulatory system genes, suggesting that the Fur protein might directly be involved in the regulation of nitrogen fixation genes. Base on this hypothesis, we will firstly try to fish out the direct targets of fur protein involved in the nitrogen regulation system by the comprehension analysis of mutant phenotype determination, transcriptome sequencing and real-time quantitative RT-PCR. Secondly, we will try to figure out the regulatory mechanism of Fur in modulating the expression of the nif genes by using macromolecules interaction analytical methods, such as EMSA and the newly developed MST technique. Finally, we will establish a working model in Pseudomonas stutzeri A1501 on how the cells responses to the external ferric signals and thereafter activates the expression of nif genes. This work may lay an important theoretical basis for the molecular mechanism of iron uptake, storage regulation in bacteria and also will highlight the regulation mechanism of nitrogen fixation systems.

革兰氏阴性菌中，铁转运、储存和利用主要由铁吸收调节蛋白Fur调控。大量研究表明，Fur在铁转运、氧化胁迫、微生物-宿主互作过程中发挥重要作用。铁是成熟固氮酶的重要组分，铁的获取及利用能力对固氮酶的功能发挥至关重要，但是Fur蛋白是否直接参与固氮调控未见报道。施氏假单胞菌A1501是研究联合固氮的模式菌株，前期研究发现该菌fur基因的突变造成固氮酶活下降90%，而且固氮酶基因nifH和固氮调控基因nifA的表达量显著下调，但一般氮代谢系统变化不明显，暗示着Fur蛋白直接参与了固氮基因的表达调控。本研究拟通过突变株表型分析、转录组测序和实时定量等方法明确Fur参与固氮系统调控的作用靶标，并通过凝胶阻滞、微量热涌动实验等生物大分子互作方法解析Fur蛋白调控固氮基因表达的分子机制，建立固氮菌响应外界铁信号开启固氮基因表达的信号传递模型，为解析微生物胞内铁平衡调节及高效固氮的分子机制奠定理论基础。

铁是细胞生理代谢的重要金属元素，在生物氧化、分解代谢、氧化胁迫反应等方面具有重要作用。但过量的铁会对细胞产生毒性，生物体必需在铁的摄取与铁过多导致的毒性之间保持平衡。革兰氏阴性菌中，胞内铁转运、储存和利用主要由铁吸收调节蛋白Fur调控。铁是成熟固氮酶的重要组分，铁的获取及利用能力对固氮酶的功能发挥至关重要。前期研究发现，施氏假单胞菌A1501中Fur的突变造成固氮酶活下降90%，固氮酶基因下调显著，但一般氮代谢系统变化不明显，暗示着Fur蛋白直接参与了固氮基因的表达调控。.本研究聚焦A1501菌中Fur蛋白调控固氮基因表达的分子机制开展工作。首先通过实时定量和Western杂交的方法明确了fur基因的表达特征，发现A1501菌fur基因在限铁条件下高水平表达并受一般胁迫sigma因子RpoS的调控； fur基因突变株的功能分析表明，Fur参与了A1501菌的胞内铁离子转运和固氮基因表达调控，对铁转运基因表达存在负调控，对固氮基因的表达具有正调控作用；分别测定了fur突变株和野生型A1501在不同培养条件下的转录组，绘制了fur基因的全局调控网络，并结合生物信息分析以及蛋白-DNA互作鉴定了一系列Fur的作用靶标；凝胶阻滞实验证明Fur蛋白与nifHDK的启动子以及prrF的启动子存在体外结合现象，表明Fur蛋白对nifH和prrF的表达存在直接调节作用；RNA二级结构分析发现PrrF与nifH和nifQ mRNA存在可能结合位点，并通过MST实验证实了PrrF与两mRNA均存在体外互作；综合以上研究结果，绘制了A1501菌中Fur蛋白直接参与固氮基因活性调控的分子模型：在转录水平，Fur蛋白与固氮酶铁蛋白基因nifH的启动子互作，正调控nifH基因的表达；在转录后水平，Fur蛋白负调控ncRNA PrrF的表达，PrrF通过与nifH mRNA及钼铁辅因子合成基因nifQ mRNA直接互作，影响固氮酶的合成及组装。.项目圆满完成考核指标，获得了一系列原创性研究成果，已发表学术论文15篇，其中SCI 收录8篇，中文核心期刊7 篇；项目执行期间参加国际、国内学术交流9次；指导博士研究生4名，硕士研究生5 名。本研究首次发现铁转运蛋白在转录和转录后水平直接参与固氮调节的分子机制，为深入解析固氮菌的调控网络及环境适应机制奠定了理论基础。