类芽孢杆菌固氮基因簇在大肠杆菌和枯草芽孢杆菌中的表达和调控研究

Nitrogen fixation has been studied extensively in various microorganisms. The well studied nitrogen-fixing microorganisms are Gram-negative bacteria including Klebesiela pneumoniae, Pseudomonas stutzeri, Azotobacter vinelandii, Azospirillum brasilense and Gram-positive bacteria including Heliobacterium chlorum.The organization of nitrogen fixation (nif) genes has great difference in Gram-negative and Gram-positive bacteria. In K. pneumoniae, 21 adjacent nif (nitrogen fixation) genes are organized in eight operons (nifJC, nifHDKTY, nifEN, nifUSVW, nifZM, nifF, nifLA, nifBQ) within ca. 24 kb of DNA. All nif genes are transcribed from σ54 promoters (-24/-12). The nifD and nifK genes specify the α and β subunits, respectively, of the molybdenum iron protein. The iron protein is encoded by nifH. The nifEN and nifB are required for the synthesis of FeMo-co factor of nitrogenase. NifA, the transcriptional activator, is required for expression of other nif genes. When the nif cluster is introduced to Escherichia coli, the E. coli has nitrogenase activity. However, the nitrogenase activity is affected by ammonium and oxygen.. Recent research shows that the minimal genes for nitrogen fixation are the 6 genes： nifH nifD nifK nifB nifE nifN.Our research results show that nitrogen fixation in Gram-positive Panebacillus sabinae T27 and Panebacillus beijingnica WLY1-18 is governed by a nif cluster consisting of 8 genes nifBHDKENXV.However, the identity between the 2 nif clusters is only 70%,suggesting that the 2 nif clusters might show difference in nitrogen-fixation..In this study, the work will be completed as follows: (1) to complete the genome sequence of P. beijingnica WLY1-18.(2)To determine the transcription origin and transcription unit of the nif cluster in P. beijingnica WLY 1-18和P. sabinae T27.(3)To clone the nif cluster of nifBHDKENXV from P. beijingnica WLY 1-18 and P. sabinae T27, and then the nif clusters will be under the control of nif-promoter and lacZ promoter. (4)To transfer the nif clusters to Escherichia coli and Bacillus subtilis, respectively. (5)To check the nitrogenase actitivy of the recombinant E. coli and B. subtilis.(6)To determine the nitrogenase level in E. coli and B. subtilis by Western blot. (7)To determine the effects of ammonium and oxygen on expression of nifH nifD nifK in the recombinant E. coli and B. subtilis by Quantitative Real-Time RT-PCR.

美国最近用体外固氮酶研究表明，生物固氮只有6个必需基因nifHDKBEN。但国内外尚没有证明6个固氮基因能否在非固氮菌，如大肠杆菌中完成生物固氮作用。我室从固氮类芽孢杆菌Paenibacillus sabinae和Paenibacillus beijingnica中各自克隆到1个由8个基因组成的固氮基因簇nifHBDKENXV，这2个固氮基因簇在大肠杆菌中有无固氮作用？固氮作用受铵和氧调控吗？为回答这些问题，本研究内容为：（1）完成P. beijingnica全基因组序列分析；（2）确定2个固氮基因簇转录起始位点和转录单元；（3）将2个固氮基因簇分别在自身启动子和lacZ启动子带动下，在大肠杆菌和枯草芽孢杆菌表达；（4）重组大肠杆菌和枯草芽孢杆菌固氮酶活性测定；（5）铵和氧对固氮基因在重组大肠杆菌和枯草芽孢杆菌的表达调控研究。结果将为构建高效固氮工程菌株和扩大寄主范围及转基因植物奠定基础

类芽孢杆菌（Paenibacillus）是一类能产生芽孢的革兰氏阳性细菌。我们完成了13个固氮类芽孢杆菌基因组序列分析，发现固氮类芽孢杆菌中均存在一个由9个基因（nifB H D K E N X hesA nifV）组成的固氮基因簇，是目前发现的结构最紧密的、最小固氮基因簇之一。从固氮多粘类芽孢杆菌（P. polymyxa）WLY78克隆9个基因组成的最小固氮基因簇（nifBHDKENXhesAnifV）及其启动子（11kb），导入大肠杆菌JM109获得的重组大肠杆菌78-7具有固氮能力；基因缺失研究发现，9个固氮基因能维持最高固氮酶活性，只有6个固氮基因时没有任何固氮活性。9个基因具有固氮活性这一研究发现，具有划时代意义，因为模式固氮菌---肺炎克氏菌完成生物固氮至少需要16个基因。. 利用RT-PCR方法确定了P. polymyxa WLY78中9个固氮基因（nifBHDKENXhesAnifV）组成一个转录单元，并利用5‘RACE方法确定了该固氮基因簇的转录起点。首次用凝胶阻滞（EMSA）和Footprinting研究表明类芽孢杆菌中nif基因启动子属于σ70型（-35,-10），这与大多数革兰氏阴性固氮菌nif基因启动子是σ54型（-12,-24）完全不同。转录组分析表明，P. polymyxa WLY78中的nif基因在非固氮条件下（100 mM铵和21%氧气）不表达，而在固氮条件下（无铵和无氧）高水平表达，上调表达水平达1000倍，说明与革兰氏阴性固氮菌类似，nif基因在革兰氏阳性的类芽孢杆菌中的表达也受铵和氧调控。但重组大肠杆菌78-7（携带来自P. polymyxa WLY78的固氮基因簇）在固氮和非固氮条件下均高水平表达，说明固氮基因在大肠杆菌摆脱了铵和氧的抑制。. 将 9个固氮基因（nifBHDKENXhesAnifV）导入到枯草芽孢杆菌中， 枯草芽孢杆菌也获得了固氮能力。