GlnR 在类芽孢杆菌固氮基因表达中的调控作用

Nitrogen fixation is highly energy expensive and is regulated tightly both transcriptionally and post-translationally according to ammonium concentration. Our previous results with RT-PCR analysis showed that the transcription of nif (nitrogen fixation) gene of Paenibacillus polymyxa WLY78 is affected by ammonium. However, the regulation mechanisms of nitrogen fixation by ammonium in Paenibacillus is not known. Our recent studies have shown that there is a complete nif cluster composed of 9 genes nifBHDKENXhesAnifV in P. polymyxa WLY78.RT-PCR showed that the 9 nif genes are organized as an operon.The nif promoter (-35/TTGACT and -10 /TAAGAT) is similar to the σ70 promoter (-35 TTGACA and -10/ TATAAT) of Escherichia coli, which is very different from the σ54 promoters (-24/-12) of nif genes in the Gram-negative bacteria, such as Klebesiela pneumoniae, whose nif gene expression requires the activation of the transcriptional activator NifA according to the concentration of ammonium. Importantly, we discovered a GlnR-binding site (TGTAACCGCGCACA) in the nif promoter of P. polymyxa WLY78. In the Gram-positive model organism Bacillus subtilis, two transcriptional factors, TnrA and GlnR, control gene expression in response to nitrogen availability. TnrA activates and represses gene transcription when nitrogen is limiting for growth, while GlnR represses gene expression during growth with excess nitrogen. In order to understand the regulatoion mechnisms of nitrogen fixation by ammonium in Paenibacillus, the following research work will be done: (1) To clone glnR gene from P.polymyxa WLY78 and then it is constructed to expression vector pET-28b and expressed in E. coli BL21. The GlnR protein will be purified from E. coli BL21.(2) The GlnR protein will be used to bind the nif promoter of P. polymyxa WLY78 by using Electrophoretic mobility shift assay (EMSA）and Footprinting assay. (3) To co-transfer the glnR and nif gene cluster from Paenibacillus sp. WLY78 to E. coli JM109. Then the effects of glnR on expression of the nif gene cluster under different concentration of ammonium in E. coli will be studied by real-time RT-PCR. (4) To construct ΔglnR mutant, the partial glnR gene will be ligated to vector pBluescript SK(+) and then trnasformed to P. polymyxa WLY78 and ΔglnR mutant will be selected. (5) To analyze the expression of nifB nifH nifD nifK nifE nifN nifX hesA nifV in both P.polymyxa WLY78 and the ΔglnR mutant by real-time RT-PCR under excess ammonium and without ammonium conditions. (6)To determine which promoters are bound by GlnR by using Chromatin Immunoprecipitation (ChIP) techenique.(7) To implement the transcriptomic analysis of P. polymyxa WLY78 and the ΔglnR mutant under conditions of excess ammonium and without ammonium to determine which genes are up-regulated and down-regulayed.

固氮类芽孢杆菌（Paenibacillus）抗逆力强和存活期长，是制备生物肥料的最佳菌种。与其它固氮菌类似，固氮类芽孢杆菌的固氮效率受铵调控，但其固氮调节机理尚不清楚。最近我室研究发现，类芽孢杆菌的的固氮基因（nif）启动子属于σ70型，启动子内有GlnR蛋白结合位点，不同于大多数nif启动子属于σ54型，其转录严格依赖NifA蛋白。本项目研究目标是确定 GlnR根据铵浓度调控nif及氮代谢基因的表达。研究内容主要是：1、凝胶阻滞和Footprinting确定GlnR蛋白与nif启动子结合；2、ΔglnR 突变株构建；3、利用qRT-PCR研究在高铵和低铵条件下，野生型类芽孢杆菌和ΔglnR株中nif基因的表达；4、染色质免疫沉淀技术研究体内GlnR与哪些基因启动子结合；5、全基因组转录分析确定哪些基因受GlnR调控;6、研究异源大肠杆菌中，GlnR对nif基因的调控作用。

1. 项目研究背景. 固氮微生物在常温常压下,利用体内的固氮酶能将空气中的氮气还原成铵，供植物生长利用。但固氮效率受铵浓度的影响，即，在无铵或铵浓度低时固氮，铵浓度一般大于5 mM以上抑制固氮。在贫瘠的土壤里，固氮微生物的固氮效率高，而在肥沃的土壤里，只生长但不固氮。因此，阐明固氮调控机理，获得在高铵条件下固氮的微生物，在农业生 产中具有重要的应用价值。.2. 主要研究内容. （1）glnR基因的克隆和在大肠杆菌中的表达；（2）凝胶阻滞（EMSA）和 Footprinting 确定GlnR 与固氮基因启动子的结合；（3）GlnR 蛋白在异源大肠杆菌中对 nif 基因转录和活性的影响；（4）ΔglnR 突变株的构建；（5）ΔglnR 突变株中固氮基因的表达；(6) 利用染色质免疫沉淀技术（Chromatin Immunoprecipitation，ChIP）研究在固氮类芽孢杆菌体内GlnR蛋白质与哪些基因的启动子DNA结合。.3. 重要结果. 通过基因突变、凝胶阻滞（EMSA）、表面等离子体（SPR）、免疫共沉淀（ChIP）等研究表明，在固氮类芽孢杆菌中，氮代谢总调控因子GlnR和GS（谷氨酰胺合成酶）调控固氮基因的表达。我们的研究率先揭示出了一个全新的固氮调控机理。在固氮基因簇（nifBHDLENXhesAnifV）上游的启动子区，有两个GlnR结合位点，分别位于启动子-35/-10区上下游。在无铵条件下，GlnR与上游位点结合激活固氮基因表达；在高铵条件下，GlnR与下游位点结合抑制固氮基因表达。谷氨酰胺是氮的信号传递者。铵浓度高时，GS催化铵和谷氨酸合成谷氨酰胺，谷氨酰胺反馈抑制GS、并与GS相互作用形成反馈抑制型谷氨酰胺合成酶（FBI-GS），FBI-GS参与GlnR抑制固氮基因转录。这是首次阐明固氮类芽孢杆菌中独特的固氮调控机理。同时，对拓展研究GlnR同时具有激活和抑制调控作用也具有重要的意义，因为在枯草芽孢杆菌等阳性细菌中，GlnR被认为在氮代谢中只起抑制作用。另外，该研究表明，对编码GS的glnA基因缺失、对编码GlnR的glnR基因增加拷贝数及对GlnR的第2个结合位点进行突变，都能使固氮微生物在有铵（0-400 mM）条件下固氮，在农业生产中具有重要的应用价值。