pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Cloning of pEA3, a large plasmid ofEnterobacter agglomerans containing nitrogenase structural genes

Summary A clone bank of an indigenous plasmid ofEnterobacter agglomerans containing structural nitrogen-fixation (nif) genes was established in a non-mobilisable, multicopy derivative of the cosmid vector pHC79. The restriction enzyme Bam HI was used to establish the clone bank and it was found that 96% of the clones contained inserts. The clones containingnif-genes were identified by Southern hybridisation usingKlebsiella pneumoniae nif DNA (KpnifHDKY) as the radioactive probe. Thenif-genes ofE. agglomerans showed extensive homology to those ofK. pneumoniae but the restriction enzyme fragment patterns of thenif-genes ofE. agglomerans were different. The plasmid bornenif-genes ofE. agglomerans are clustered as inK. pneumoniae.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

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。     

Site-specific mutadenesis in Enterobacter agglomerans: construction of nifB mutants and analysis of the gene's structure and function

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB ^– mutants with the kanamycin cassette inserted in either orientation showed sequence of nifb. A typical ⁵⁴-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.     

Cluster analysis of genes for nitrogen fixation from several diazotrophs

Hierarchical clustering and similarity coefficients of pairwise alignments of the published nucleotide sequences of 27nifH genes suggest thatnif genes are as ancient as the archaebacteria and clostridia. The positions ofnifHl ofMethanococcus thermolithotrophicus, nifH3 ofClostridium pasteurianum, nifH3 ofAzotobacter vinelandii andnifH ofFrankia suggest that a variety of lateral transfers may have occurred during evolution ofnifH gene. The genes for type 3 nitrogenase ofA. vinelandii may have diverged early from methanogens and clostridia. A high similarity coefficient with the derived amino acid sequence of type 3 nitrogenase suggests the presence of a functionally similar enzyme inC. pasteurianum. The type 2 nitrogenase genenifH2 of azotobacters seems to have originated recently from the genenifHl for conventional type I nitrogenase. RhizobialnifH genes comprise two closely related but discrete clusters that are in consonance with the plasmid or chromosomal location ofnif genes. The chromosomal and plasmid locatednifH of rhizobia seem to have evolved independently but contemporaneously.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA ^pro gene), proteolysis (pcr genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation

Summary The plasmid pattern of Rhizobium meliloti strain GR4 was studied and a gene bank of one of the large plasmids (pRmeGR4) of 140 Mdal, was constructed using the broad host range vector pRK290. A restriction map was established with EcoRI. Two regions of this plasmid involved in the infectivity of GR4 on Medicago sativa were identified. An EcoRI fragment hybridizing with the PstI-nif fragment of pID1 was also identified. However, no homology to the cloned Klebsiella pneumoniae nitrogenase genes (pSA30) was detected.     

Diversity of nif gene location and nitrogen fixation among root-associated Enterobacter and Klebsiella strains

Our previous work showed that strains of dinitrogen fixing enterobacter and Klebsiella were found associated with the roots of uncultivated grasses in Finland more commonly than other species of diazotrophic bacteria. In this paper we compare E. agglomerans strains to K. pneumoniae and K. terrigena strains, and show that the E. agglomerans strains fall into two biogroups. The groups differ not only in the utilization of different carbon sources and other physiological characteristics such as the production of indole, but also in the physiology and genetics of nitrogenase activity. Biotype 1 (isolated from Achillea millefolium, Calamagrostis arundinacea, and Phleum pratense) showed active nitrogenase in atmospheric oxygen, whereas biotype 2 (from Phalaris arundinacea) resembled K. pneumoniae in that it was active at reduced oxygen pressure (pO₂<-0.002) only. DNA of all strains showed positive hybridization with K. pneumoniae nifHDK genes (pSA30) but differed in the location of the genes. Biotype 1 strains of E. agglomerans carried nifHDK genes on large (105–125 Mdal) plasmids, whereas no plasmid was detected in biotype 2 or in the K. pneumoniae strains isolated from Agrostis stolonifera and Poa pratensis and K. terrigena strain isolated from Carex pallescens. The one K. terrigena strain (isolated from Ph. arundinacea) that was found to contain an indigenous plasmid (80 Mdal) did not carry nifHDK genes on this plasmid.     

Expression of klebsiella his and nif genes in serratia marcescens,Erwinia herbicola and Proteus mirabilis

Plasmid pRD1, an R plasmid of the P incompatibility group which carries his and nif genes from Klebsiella pneumoniae in addition to drug resistance markers derived from RP4, was transferred to His^- mutants of Serratia marcescens, Erwinia herbicola and Proteus mirabilis. His⁺ transconjugants were obtained at low but different frequencies according to recipient genus. Transconjugants all acquired the drug resistance, and were Nif⁺ in S. marcescens and E. herbicola, having acetylene-reducing activities of the same order of magnitude as the parent K. pneumoniae and fixing ¹⁵N₂. No evidence for nif expression in P. mirabilis transconjugants was obtained though the nif genes were present.     

Cre-LoxP系统在炭疽芽胞杆菌基因敲除中的应用及eag基因的敲除

将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因．以B.anthracis基因组为模板扩增得到上下游同源臂,联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体,转化B．anthracis AP422,通过一系列筛选得到带有抗性标记的重组菌．然后,通过转入Cre重组酶表达质粒,去除抗性标记,得到eag基因缺失的重组菌,并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定．最终建立了Cre-LoxP系统在B．anthracis中的应用方法,并成功敲除eag基因.     

Identification of a regulatory nifA type gene and physical mapping of cloned new nif regions of Azospirillum brasilense

Summary Three new Tn5-mutagenized nif genes of Azospirillum brasilense were characterized. The sizes of the restriction fragments and the restriction maps of the cloned nif DNA regions showed that these nif genes are distinct from those reported earlier, e.g. nifHDK, nifE, nifUS, fixABC. The Nif27 mutant was identified as a nifA type regulatory gene of A. brasilense (a) by genetic complementation with nifA of Klebsiella pneumoniae, (b) by the absence of nitrogenase iron protein in western protein blots and (c) by its inability to activate expression of a nijH-lacZ fusion. The growth characteristics of the three mutants showed that none of them is defective in general nitrogen regulatory (ntr) genes. Also, no homology was detected between the three nif DNA regions of the mutants, cloned in pMS188, pMS189 and pMS197, and the K. pneumoniae nif, gInA or ntr genes. In addition, the fixABC genes of Bradyrhizobium japonicum did not show any hybridization with the cloned Azospirillum genes. Unlike the situation in enteric bacteria, the nif genes in A. brasilense are scattered and span a region of about 65 kb.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Klebsiella pneumoniae nif-lac fusions are expressed in Agrobacterium tumefaciens C58

Summary Plasmids containing hybrid genes, in which different Klebsiella pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce -galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nif A is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of nif promoters in A. tumefaciens are discussed.