第二固氮系统在蓝细菌红萍鱼腥藻中的发现

人们一般认为金属钼是生物固氮必需的条件,钼是固氮酶的主要催化活性的组成部分。早先一些学者曾研究以钒、锰代替钼的氮固定作用,到1980年美国的Bishop等人首先提出棕色固氮菌(Azotobacter vinelandii)具有不含铝的第二固氮系统。通过棕色固氮菌缺失编码固氮酶蛋白基因突变株研究发现,该菌株并不失去     

浑球红假单胞菌GOGAT缺失株中依赖Z—酮戊二酸的固氮酶诱导

2—酮戊二酸加于限量氮培养基中后,引起泽球红假单胞菌谷氨酸合成酶(GOGAT)缺失菌株(asm~-,Nif~-)固氮酶的诱导表达,诱导生成的固氮酶的活性随着培养时间的延长而下降。此时如果加入谷氨酸,固氮酶活性又重新出现,而谷氨酸通常是阻遏GOGAT缺失菌株固氮酶表达的。诱导出的固氮酶与野生菌株固氮酶有相同的调节方式,但前后两次出现的固氮酶活性在氨的敏感性上有差异。此外,在GOGAT缺失菌株内含有较高的谷氨酰胺库,同时谷氨酸胺合成酶(GS)的腺苷酸化状态也较野生型菌株高。     

光合细菌Rhodobacter sphaeroides glnB-lacZ融合子的构建与表达

亚克隆了Rhodobacter sphaeroides glnB启动子,以pMP220为载体构建成glnB-lacZ融合子。将glnB-lacZ、 nifH-lacZ、 nifA-lacZ分别导入R.sphaeroides谷氨酸合酶突变株gltB-、 gltD-和野生型菌株中,分析了突变对固氮基因转录表达的影响。试验证明,在gltBD突变株中nifH的表达受阻遏,nifA表达水平很低。这证明glt基因的突变引起固氮酶结构基因和固氮正调节基因的转录被阻遏,而glnB基因的表达几乎不受影响。试验还测定了环境中结合态氮和有机酸等信号分子对glnB和nifH表达的影响,发现加入氨或谷氨酰胺后,nifH的表达受到明显的阻遏作用,glnB-lacZ的β-半乳糖苷酶活性虽下降30%左右但不随结合态氮浓度升高而变化,仍维持在一个较高的水平。α-酮戊二酸和丙酮酸对nifH的表达有部分去阻遏作用而对glnB的表达无诱导作用。     

大豆根瘤菌固氮酶的铁蛋白基因

大豆根瘤菌(Rhizobivmjaponicum)的固氮酶具有与肺炎克氏杆菌(Klebsiella Pneumoniae)和苜蓿根瘤菌(R.meliloti)二者固氮酶相同的铁蛋白基因(nif H)。铁蛋白基因的大小为12.1Kb,部分基因片段已经克隆化。将nifH的一部分克隆化,并使之在大肠杆菌(E.Coli)     

自生条件下根瘤菌固氮酶活性的表达

在自生条件下,测定从四种热带豆科植物中分离得到的七株根瘤菌乙炔还原能力,获得四株根瘤具有固氮活性。南洋楹菌株8638L、8638M、8638S和四棱豆菌株pS,其中菌株8638L和pS表现较高的固氮活性。结果表明,在培养基中含有低浓度氮源(谷氨酰胺)、两种碳水化合物(阿拉伯糖和琥珀酸钠)及低浓度的氧,是根瘤菌表达较高固氮酶活性所必需的。菌株8638L和pS固氮酶表达最适氧浓度为1％,谷氨酰胺浓度分别为2mMol/L     

野大豆多功能根际促生菌的筛选鉴定和促生效果研究

为获得野大豆根际促生菌(Plant growth promoting rhizobacteria,PGPR)并明确其促生特性,采用选择培养方法从沈阳沈北新区蒲河沿岸生长的野大豆根瘤及根际土壤中分离筛选固氮菌与解磷菌,进行形态学、生理生化指标和16S rDNA分子生物学鉴定。采用Touch-Up PCR(上升PCR)的方法扩增固氮酶nifH结构基因,对菌株的解磷、分泌3-吲哚乙酸(IAA)的能力及ACC脱氨酶的活性进行测定分析,筛选出优良菌株进行盆栽大豆的促生作用研究。结果得到12株野大豆PGPR菌,经鉴定分别归属于芽孢杆菌属(Bacillus)、伯克霍尔德菌属(Burkholderia)、根瘤菌属(Rhizobium)、詹森氏菌属(Janthinobacterium)、鞘氨醇单胞菌(Phingomonas)、分枝杆菌属(Mycobacterium)、假单胞菌属(Pseudomonas)和根癌农杆菌属(Agrobacterium);其中2株可解有机磷,11株可解无机磷,3株具分泌IAA能力,5株具ACC脱氨酶活性。盆栽试验表明:3株菌(GD11、GD17、GD58)可使大豆幼苗株高增长,5株菌(GD9、GD11、GD17、GD30、GD58)可使根长增长;6株菌可提高大豆幼苗的茎干重、叶干重、根干重。野大豆根瘤和根际土壤含有多功能根际促生菌,其在农业生产上具有潜在的应用价值。     

GenoType MTBDRplus在内蒙古地区耐多药结核菌检测中的应用评价

从2001年WHO/IUATLD全球抗结核药物耐药监测之内蒙古耐药监测结核菌1 114株中,选取经比例法药敏结果得到的耐多药(MDR)结核菌188株,利用Geno Type MTBDRplus方法检测该批菌株的RIF、INH耐药情况及其耐药基因突变形式。最终得到MDR菌株103(54.79%)株,单耐RIF菌株52(27.66%)株,单耐INH菌株10(5.32%)株,全敏感菌株20株,TUB条带缺失1株,kat G质控带缺失2株。RIF耐药菌株检测的是rpo B基因区,该基因突变菌株有155株;rpo B S531L突变菌株为49.68%(77/155)。INH耐药菌株检测的是kat G基因区和inh A基因启动子区,共113株,其中kat G基因突变菌株占79.65%(90/113),主要是S3-15T1突变;inh A基因突变菌株占22.12%(25/113)。因此,Geno Type MTBDRplus可用于内蒙古地区MDR结核菌的快速检测。其中rpo BS531L突变形式在RIF耐药菌株中最常见;在INH耐药菌株中,kat G基因突变较inh A基因突变常见,S315T1突变是INH耐药菌中常见的突变形式。     

念珠藻属植物nifH基因的适应性进化分析

念珠藻(Nostoc)固氮过程关键在于固氮酶的催化,而固氮酶复合物中的铁蛋白(NifH)是由高度保守的nifH基因编码的,该基因是进化史上现存最古老的功能基因之一。该研究选取念珠藻属及近缘类群的nifH基因序列共40条,采用最大似然法构建系统发育树;运行PAML4.9软件,对nifH基因编码蛋白进行生物信息学分析,并使用分支模型、位点模型和分支-位点模型检测该基因的选择位点,探讨nifH基因的适应性进化特征。结果表明:(1)最大似然树显示内类群中该研究物种共分为6个分支(A、B、C、D、E和F),其中D和E是2个大的分支,每个大分支中又各包含2个特殊的小分支A、F和B、C,其中F分支包含新疆古尔班通古特沙漠采集到的9株念珠藻,A分支包含F分支及该研究测定序列的4株葛仙米,B分支包含本研究测定序列的4株地皮菜和3株未定种的念珠藻,C分支包含NCBI数据库中下载的5株念珠藻、鱼腥藻序列和本研究测定序列的1株念珠藻。(2)在所分析的3种进化模型中,仅通过分支-位点模型检测出14个统计学上显著的正选择位点,即1F、2S、3S、4T、5A、6F、7F、8I、9S、10C、17I、27Y、29D和31R位点,表明念珠藻属植物的nifH基因发生了适应性变化,分支-位点模型是研究藻类基因适应性进化较好的模型。     

浑球红假单胞菌及其谷氨酸合成酶突变株氢酶活性和合成

浑球红假单胞菌野生型菌株的氢酶表达被有机碳、氮底物所抑制。在光照和黑暗时,氧浓度变化对氢酶的作用不同,但高氧浓度都阻遏氢酶的表达。微量Ni~(2+)能专一性地促进氢酶活性,固氮酶的产氢也可以调节氢酶的表达水平。该野生菌株的GOGAT突变株缺乏固氮酶和氢酶活性,在加入谷氨酰胺合成酶抑制剂MSX后,固氮酶和氢酶以相关联的方式合成出来,固氮酶产生的氢看来诱导了氢酶的合成。然而在固氮酶不表达的情况下,外源氢也可诱导氢酶的合成。     

四株红树林促生菌的遗传分析鉴定及其促生能力

【目的】鉴定四株供试菌株的种属地位,了解菌株所具有的促进植物生长能力。【方法】运用nifH与16S rRNA基因序列对供试菌株进行遗传分析,采用钼锑抗比色法和乙炔还原法分别测定菌株的溶磷、固氮能力。通过接种试验验证菌株促进红树植物生长的能力。【结果】通过对菌株nifH与16S rRNA的同源性、系统发育树分析,HN011与需钠弧菌(Vibrio natriegens)的相似性最高,SZ7-1、SZ7-2与产酸克雷伯氏菌(Klebsiella oxytoca)的相似性最高。而SZ002在16S rRNA的系统发育分析中归属为类芽孢杆菌属(Paenibacillus sp.),却在nifH基因分析中与克雷伯氏菌(Klebsiella sp.)的相似性最高。供试菌株都具有较强的溶磷能力和高固氮酶活性。接种后植株有较好的生长表现,部分接种植株在干重、全氮、全磷含量等方面较对照有显著地增加(P0.05)。【结论】首次发现兼具溶磷-固氮两种能力的红树林植物促生菌,接种试验也表现菌株具有良好的促生能力,为红树林人工接种促生菌的应用提供了可靠的理论依据。     

The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.     

Nucleotide sequence of the R.meliloti nitrogenase reductase (nifH) gene.

The nucleotide sequence of the structural gene (nifH) of nitrogenase reductase (Fe protein) from R.meliloti 41 with its flanking ends is reported. The amino acid sequence of nitrogenase reductase was deduced from the DNA sequence. The predicted R.meliloti nitrogenase reductase protein consists of 297 amino acid residues, has a molecular weight of 32,740 daltons and contains 5 cysteine residues. The codon usage in the nifH gene is presented. In the 5' flanking region, sequences resembling to consensus sequences of bacterial control regions were found. Comparison of the R.meliloti nifH nucleotide and amino acid sequences with those from different nitrogen-fixing organisms showed that the amino acid sequences are more conserved than the nucleotide sequences. This structural conservation of nitrogenase reductase may be related to its function and may explain the conservation of the nifH gene during evolution.     

Nitrogenase activity of Herbaspirillum seropedicae grown under low iron levels requires the products of nifXorf1 genes

Herbaspirillum seropedicae strains mutated in the nifX or orf1 genes showed 90% or 50% reduction in nitrogenase activity under low levels of iron or molybdenum respectively. Mutations in nifX or orf1 genes did not affect nif gene expression since a nifH::lacZ fusion was fully active in both mutants. nifX and the contiguous gene orf1 are essential for maximum nitrogen fixation under iron limitation and are probably involved in synthesis of nitrogenase iron or iron-molybdenum clusters.     

nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Quinoprotein d-glucose dehydrogenase in Acinetobacter calcoaceticus LMD 79.41: the membrane-bound enzyme is distinct from the soluble enzyme

Abstract The hifH gene from Klebsiella pneumoniae , which codes for the Fe protein component of nitrogenase, was used as a probe to detect nifH homologues in total cellular DNA from 13 obligate methane oxidizing bacteria. All but one of those strains that had previously been shown capable of fixing dinitrogen contained sequences homologous to nifH . DNAs from three of the six non-diazotrophic strains were also found to possess such homology.     

Consortial N2 fixation: a strategy for meeting nitrogen requirements of marine and terrestrial cyanobacterial mats

Abstract: Four microbial mat-forming, non-axenic, strains of the non-heterocystous, filamentous, cyanobacterial genus Microcoleus were maintained in culture and examined for the ability to fix atmospheric nitrogen (N₂). Each was tested for nitrogenase activity using the acetylene reduction assay (ARA) and for the presence of the dinitrogenase reductase gene ( nifH ), an essential gene for N₂ fixation, using the polymerase chain reaction (PCR). The Microcoleus spp. cultures were incapable of growth without an exogenous nitrogen source and never exhibited nitrogenase activity. Attempts to amplify a 360-bp segment of the nifH gene using DNA purified from the cyanobacterial cultures did not produce any cyanobacteria-specific nifH sequences. However, several non-cyanobacterial homologous nifH sequences were obtained. Phylogenetic analysis showed these sequences to be most similar to sequences from heterotrophic bacteria isolated from a marine microbial mat in Tomales Bay (California, USA), and bulk DNA extracted from a cryptobiotic soil crust in Moab (Utah, USA). Microcoleus spp. dominated the biomass of both systems. Cyanobacteria-specific 16S rDNA sequences obtained from the cultured cyanobacterial strains demonstrate that the lack of cyanobacteria-specific nifH sequences was not due to inefficiency of extracting Microcoleus DNA. Hence, both the growth and genetic data indicate that, contrary to earlier reports, Microcoleus spp. appear incapable of fixing N₂ because they lack at least one of the requisite genes for this process. Furthermore, our study suggests epiphytic N₂-fixing bacteria form a diazotrophic consortium with these Microcoleus spp. and are likely key sources of fixed N₂ generated within soil crusts and marine microbial mats.     

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.     

Expression of nitrogen fixation genes in foreign hosts. Assembly of nitrogenase Fe protein in Escherichia coli and in yeast

In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase. Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K. pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide. This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E. coli and in yeast. Immunoblotting methods, as well as 55Fe2- labeling of K. pneumoniae were employed to detect native nitrogenase components in cell lysates. E. coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP. While in E. coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.