肺炎克氏杆菌NifA对巴西固氮 螺菌nifH启动子的转录激 作用

用ＰＣＲ方法克隆了巴西固氮螺菌Ｙｕ６２ｎｉｆＨ的启动子片段,ＤＮＡ序列分析表明菌株Ｙｕ６２与标准菌株Ｓｐ７之间的ＤＮＡ序列差异很小。利用启动子探针质粒载体ｐＣＢ１８２,构建了３个不同的ｎｉｆＨ１ａｃＺ转录融合质粒,在大肠杆菌中分别测定肺炎克氏杆菌ＮｉｆＡ对它们的转录激活作用。结果表明巴西固氮螺菌ｎｉｆＨ启动子的转录是依赖于ＮｉｆＡ的,缺失了上游激活序列的启动子不能被ＮｉｆＡ激活转录,肺炎克氏杆菌Ｎ     

巴西固氮螺菌中PⅡ和Pz在固氮调节中的不同使用

在巴西固氮螺菌（Azospirillum brasilense)中,glnB和glnZ是两个高度同源基因,分别位于3.7kgb/EcoRI PstI和3.7kb/SalI的两个不同的染色体片段上。用卡那霉素盒（Km^r-cas-sette)插入法,对glnB和glnZ分别进行定位诱变,并获得相应的突变株,即glnB^-和glnZ^-。研究表明,glnB^-突变株丧失固氮酶活性,表现为Nif^-,glnZ^-象野生型菌株一样具有固氮酶活性。为了进一步研究这两个基因的功能,将glnB和glnZ分别构建在pVK100载体上形成重组质粒pVK-Ⅱ和pVK-Z,对glnB^-和glnZ^-突变株进行互补实验,进一步证明了glnB与固氮酶活直接相关性,而glnZ无此作用。同时,通过三亲接合法将pVK-Ⅱ和pVK-Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT^-突变株中,使glnB和glnZ的拷贝数增加,进一步比较它们的固氮酶活性。结果表明多拷贝的glnB基因,能显著提高固氮酶活性,而多拷贝的glnZ对固氮酶活性无影响。同时,将pVK-Ⅱ和pVK-Z分别转移到nifA^-突变株中,结果表明glnB和glnZ均不能恢复nifA^-的固氮酶活性。     

巴西固氮螺菌中吸氢酶基因同源性的分子检测

通过TTC(2 ,3,5 氯化三苯基氮唑 )实验筛选出 12株能产生吸氢酶蛋白 (Hup+ )的巴西固氮螺菌 (Azospir rilumbrasilense)菌株。用Qiagen柱分离提纯含豌豆根瘤菌的hup基因片段的质粒 pHVT10 9和pHVT115 ,并用地高辛标记法标记pHVT115 ,与 12株Hup+ 巴西固氮螺菌的总DNA进行斑点杂交 ,结果显示pHVT115所含的吸氢酶基因 (hup基因 )片段与巴西固氮螺菌的大部分菌株的hup基因同源性不强。这一结果表明hup基因在固氮生物中存在着遗传多样性 ,异源hup基因探针不一定都适宜于探测hup基因。     

巴西固氮螺菌中P_Ⅱ和P_Z在固氮调节中的不同作用

在巴西固氮螺菌 (Azospirillumbrasilense)中 ,glnB和glnZ是两个高度同源基因 ,分别位于 3 7kb EcoRI+PstI和 3 7kb SalI的两个不同的染色体片段上。用卡那霉素盒 (Kmr cas sette)插入法 ,对glnB和glnZ分别进行定位诱变 ,并获得相应的突变株 ,即glnB- 和glnZ- 。研究表明 ,glnB- 突变株丧失固氮酶活性 ,表现为Nif- ,而glnZ- 象野生型菌株一样具有固氮酶活性。为了进一步研究这两个基因的功能 ,将glnB和glnZ分别构建在pVK1 0 0载体上形成重组质粒pVK -Ⅱ和pVK -Z ,对glnB- 和glnZ- 突变株进行互补实验 ,进一步证明了glnB与固氮酶活有直接相关性 ,而glnZ无此作用。同时 ,通过三亲接合法将pVK -Ⅱ和pVK -Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT- 突变株中 ,使glnB和glnZ的拷贝数增加 ,进一步比较它们的固氮酶活性。结果表明多拷贝的glnB基因 ,能显著提高固氮酶活性 ,而多拷贝的glnZ对固氮酶活性无影响。同时 ,将pVK Ⅱ和pVK -…     

固氮螺菌与植物的相互关系研究进展

固氮螺菌与植物的相互关系研究进展阎大来,何路红,李季伦（北京农业大学农业生物技术国家重点实验室,北京１０００９４）固氮螺菌（Ａｚｏｓｐｉｒｉｌｌｕｍ）首先是从巴西热带禾本科牧草俯仰马唐（Ｄｉｇｉｔａｒｉａｄｅｃｕｍｂｅｎｓ）根际分离得到的￣［１,２］...     

巴西固氮螺菌Yu62nifA基因克隆、测序及功能分析

用TD-PCR法克隆了巴西固氮螺菌(Azospirillun brasilense)Yu62的nifA基因.序列分析表明它与巴西固氮螺菌Sp7的nifA序列高度同源(96.5%),其编码的产物NifA蛋白与Sp7菌株NifA的氨基酸序列同源性为97.6%.该基因可以完全互补巴西固氮螺菌Sp7 nifA-突变株的Nif-表型.研究了NH4+和O2对Yu62 nifA基因的表达及NifA活性的影响.结果表明mfA基因在Yu62菌株中是部分组成型表达的,氨和氧不能完全阻遏其表达,在5mmol/LNH4Cl与微氧(0.5%O2)条件下表达最高;NifA蛋白在0.4%～0.5%O2时活性最高,氧分压降低和提高都使NifA活性下降,1mmol/L NH4Cl足以抑制NifA的活性.     

肺炎克氏杆菌的nifA基因产物在巴西固氮螺菌中的功效

通过三亲本杂交将质粒pCK3{携带改变了启动子的肺炎克氏杆菌(Klebsiella pneuma-niae)nifA 基因]引入巴西固氮螺菌(Azospirillum brasilense)Yu62菌株中,由此获得的转移接合子巴西固氮螺菌Yu62-4菌株在6．0 mmol/L以上NH+4浓度下,能表现出微弱的固氮酶活性(相当于无NH+4时活性的0．3-0．5％),而野生型Yu62则全部丧失固氮酶活性。固氯酶的丙烯酰胺凝胶电泳和铁蛋白的免疫杂交实验表明,转移接合子Yu62-4在高NH+4(50mmol／L)下,虽有铁蛋白合成,但合成量比无NH+4时少得多,而且有一部分铁蛋白未被共价修饰;野生型菌株Yu62在此NH+4浓度下无铁蛋白合成。实验结果表明：外源(来自肺炎克氏杆菌)的基因产物在巴西固氮螺菌Yu62中不能有效地解除NH+4对该菌固氮酶合成的阻遏作用。本文分析了出现这种现象的原因。     

肺炎克氏杆菌nifA基因在巴西固氮螺菌固氮基因表达的铵调节中的作用

固氯螺菌(Azospirillum)是一类仅在限铵和微好氧条件下固氮的微生物,它可与许多禾本科作物联合共生⑴,具有较大的应用潜力。铵作为固氮作用的调节信号,在固氮螺菌的实际应用中是首要的限制因素。在固氯螺菌中,铵不但具有与肺炎克氏杆菌(Klebsiella pneumoniae)相似的阻遏固氮酶合成的作用,而且还对已合成的固氮酶进行活性调节⑵。研究表明,其固氮酶翻译后活性调节的机制类似于深红红螺菌(Rhodospirillum rubrum)⑶,即在有铵条件下其固氮酶铁蛋白的一个亚基被共价修饰而丧失活性,这一过程是可逆的。由于铵在固氮螺菌中双水平地调节固氮作用,使得在野生菌株中研究其固氮基因表达水平上的调节较为困难。Zhang等⑷利用区域定位诱变技术获得了巴西固氮螺菌Sp7（A．Brasilense Sp7)的draT-突变株,在该突变株中铵不再影响固氮酶的活性,这为其固氮基因表达调节的研究提供了一个良好的材料。本文将组成型表达的肺炎克氏杆菌nifA基围引入该突变株中,通过分析讨论铵对巴西固氮螺菌固氮基固表达的调节作用方式。     

Sequencing and complementation analysis of the nifUSV genes from Azospirillum brasilense

The functionality of nitrogenase in diazotrophic bacteria is dependent upon nif genes other than the structural nifH, D, and K genes which encode the enzyme subunit proteins. Such genes are involved in the activation of nif gene expression, maturation of subunit proteins, cofactor biosynthesis, and electron transport. In this work, approximately 5500 base pairs located within the major nif gene cluster of Azospirillum brasilense Sp7 have been sequenced. The deduced open reading frames were compared to the nif gene products of Azotobacter vinelandii and other diazotrophs. This analysis indicates the presence of five ORFs encoding ORF2, nifU, nifS, nifV, and ORF4 in the same sequential organization as found in other organisms. Consensus σ⁵⁴ and NifA binding sites are present in the putative promoter region upstream of ORF2 in the A. brasilense sequence. The nifV gene of A. brasilense but not nifU or nifS complemented corresponding mutants strains of A. vinelandii.     

Potential roles for the glnB and ntrYX genes in Azospirillum brasilense

Three Azospirillum brasilense mutants constitutive for nitrogen fixation (Nif(C)) in the presence of NH4(+) and deficient in nitrate-dependent growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-dependent growth and NH4(+) regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was restored for NH4(+) regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH4(+) regulation of nitrogenase and nitrate-dependent growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26 as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense.     

Plasmid transformation of Azospirillum brasilense

Abstract Plasmid transformation of the nitrogen-fixing bacterium Azospirillum brasilense is described. A modification of the method of Hanahan [1] was used to transform this bacterium with the 20-kb plasmid pRK290. The efficiency of transformation ranged from 200–1000 transformants per μg of plasmid DNA according to DNA concentration. Ca²⁺, Mn²⁺ and K⁺ were essential for competence, while Rb⁺ and hexamine cobalt(III) chloride did not appear necessary. The length and the temperature of heat-pulse during transformation affected the efficiency of transformation. The response to different numbers of plasmid molecules was linear, in the range of 0.05–1.0 μg of DNA. No transformants were obtained with pRK290 plasmid DNA linearized with Eco RI. The transformability of different strains of Azospirillum has been compared.     

Mutants of Azospirillum brasilense altered in nitrogen fixation

Abstract Four revertants with Nif⁺ phenotype obtained from asm mutants of Azospirillum brasilense have been studied in respect to nitrogenase, enzymes of ammonia assimilation and utilization of poor nitrogen sources. The results indicate that nitrogenase expression is related to the activity of glutamate synthase and to the adenylylation of glutamine synthetase; moreover, nitrogen fixation seems correlated with the activities of the enzymes involved in the utilization of poor nitrogen sources.