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PubMed2004

[Recent advances in research and application of associated nitrogen-fixation with graminaceous plants].

The category, characteristic of diazotrophs isolated from inside and/or rhizosphere of graminaceous plants in recent year and the mechanism of the promoting effects on their host plant were reviewed in this paper. The current status of application of associative nitrogen-fixation inoculants and the problems in inoculation were discussed. It was indicated that the main factors influencing the effects of inoculants include the competition of indigenous micro-organism with inoculants for nutritions and energy, difference of host plant genotypes in associative relationship, and variance of environmental conditions such as the concentration of ammonium in soil solution and the oxygen partial pressure in soil air. The trends of future research in this field were prospected, for example, to isolate and identify the high nitrogen fixing efficiency strains with wider environmental adaptability, to create associative nitrogen fixing bacteria strain which is able to bear or endure higher concentration of ammonium by gene engineering technique, to induce graminaceous plant forming root nodule for nitrogen fixation and to exert the predominance of endophytic diazotrophs.

Acetobacter↗

PubMed1981

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti.

R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRme41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41b was found. Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digest from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 2 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

Chromosome Mapping↗

PubMed2006

Quantitative exploration of the occurrence of lateral gene transfer by using nitrogen fixation genes as a case study.

Lateral gene transfer (LGT) is now accepted as an important factor in the evolution of prokaryotes. Establishment of the occurrence of LGT is typically attempted by a variety of methods that includes the comparison of reconstructed phylogenetic trees, the search for unusual GC composition or codon usage within a genome, and identification of similarities between distant species as determined by best blast hits. We explore quantitative assessments of these strategies to study the prokaryotic trait of nitrogen fixation, the enzyme-catalyzed reduction of N(2) to ammonia. Phylogenies constructed on nitrogen fixation genes are not in agreement with the tree-of-life based on 16S rRNA but do not conclusively distinguish between gene loss and LGT hypotheses. Using a series of analyses on a set of complete genomes, our results distinguish two structurally distinct classes of MoFe nitrogenases whose distribution cuts across lines of vertical inheritance and makes us believe that a conclusive case for LGT has been made.

Codon↗

PubMed1990

Nitrogen fixation genes involved in the Bradyrhizobium japonicum-soybean symbiosis.

The symbiotic nitrogen fixation genes (nif, fix) of Bradyrhizobium japonicum, the root nodule endosymbiont of soybean, are organized in at least two separate chromosomal gene clusters. These genes code for proteins of the nitrogenase complex, for proteins involved in their assembly with cofactors and for putative electron transport functions. One gene, nifA, codes for a transcriptional regulatory protein that plays a central role in the control of expression of the other genes in response to the cellular oxygen status. Only at low partial pressures of O2 will the target promoters be activated by NifA.

Amino Acid Sequence↗

PubMed1998

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense.

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Azospirillum brasilense↗

PubMed1982

Nitrogen fixation (acetylene reduction) associated with duckweed (lemnaceae) mats.

Duckweed (Lemnaceae) mats in Texas and Florida were investigated, using the acetylene reduction assay, to determine whether nitrogen fixation occurred in these floating aquatic macrophyte communities. N(2)-fixing microorganisms were enumerated by plating or most-probable-number techniques, using appropriate N-free media. Results of the investigations indicated that substantial N(2)-fixation (C(2)H(2)) was associated with duckweed mats in Texas and Florida. Acetylene reduction values ranged from 1 to 18 mumol of C(2)H(4) g (dry weight) day for samples incubated aerobically in light. Dark N(2) fixation was always two- to fivefold lower. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (7 to 10 muM) reduced acetylene reduction to levels intermediate between light and dark incubation. Acetylene reduction was generally greatest for samples incubated anaerobically in the light. It was estimated that 15 to 20% of the N requirement of the duckweed could be supplied through biological nitrogen fixation. N(2)-fixing heterotrophic bacteria (10 cells g [wet weight] and cyanobacteria (10 propagules g [wet weight] were associated with the duckweed mats. Azotobacter sp. was not detected in these investigations. One diazotrophic isolate was classified as Klebsiella.

Journal Article↗

PubMed2002

The Hfq-like protein NrfA of the phototrophic purple bacterium Rhodobacter capsulatus controls nitrogen fixation via regulation of nifA and anfA expression.

The Rhodobacter capsulatus nrfA gene product exhibits extensive similarity to the nif (nitrogen fixation) regulatory factor NrfA of Azorhizobium caulinodans and the nucleoid-associated protein Hfq of Escherichia coli. Mutational analysis revealed that, in contrast to the situation in A. caulinodans, NrfA is not essential for diazotrophic growth of R. capsulatus, but it is required for maximal growth rates with N(2) as sole nitrogen source via either molybdenum nitrogenase or the alternative nitrogenase. NrfA was shown to control N(2) fixation in R. capsulatus at the level of expression of the regulatory genes nifA1, nifA2 and anfA, encoding the transcriptional activators of all the other nitrogen fixation genes.

Amino Acid Sequence↗

PubMed1987

In vitro transcription of the nitrogen fixation regulatory operon nifLA of Klebsiella pneumoniae.

In vitro transcription from the promoter for the nitrogen fixation regulatory operon nifLA of K. pneumoniae requires four protein fractions: the core form of RNA polymerase; NTRA, an alternate sigma factor; NTRC, an auxiliary DNA-binding protein; and NTRB, a bifunctional enzyme that controls the activity of NTRC by covalent modification (A.J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. USA 83:5909, 1986). Two DNA-binding sites for NTRC lie approximately 150 base pairs upstream of the nifLA promoter.

Bacterial Proteins↗

PubMed1989

The Rhizobium meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed with nifA and nifB.

Sequencing of the Rhizobium meliloti DNA region downstream of nifA revealed the existence of nifB, fdxN and ORF3. The molecular weight of the fdxN protein (Mr 6830) and the distribution of cysteine residues in its deduced amino acid sequence is typical for low molecular weight bacterial ferredoxins. Interposon insertion and plasmid integration mutagenesis demonstrated that FdxN is essential for nitrogen fixation in R. meliloti, whereas the predicted translation product of ORF3 (Mr 8708) is not necessary for this process. In contrast, ferredoxin-like proteins, which are encoded by nifB-associated genes, are not required for nitrogen fixation in all other organisms analysed so far. Plasmid integration mutagenesis additionally revealed that nifA, nifB and fdxN form one transcriptional unit. This result was confirmed by complementation analysis of polar interposon insertion mutants of nifA, nifB and fdxN and by complementation of a non-polar nifA deletion mutant. A DNA sequence resembling a typical nif consensus promoter, which is preceded by two putative NifA-binding sites, is located in front of nifB. This nifB promoter can be activated in Escherichia coli by the nifA gene product of Klebsiella pneumoniae to the same level as that of the R. meliloti nifH promoter. In contrast, R. meliloti NifA stimulates the nifH promoter more efficiently than the nifB promoter. This low-level activation of the nifB promoter may be the reason why transcription of nifB and fdxN is initiated primarily at a promoter in front of nifA.

Amino Acid Sequence↗

PubMed1990

A nitrogen-fixation gene (nifC) in Clostridium pasteurianum with sequence similarity to chlJ of Escherichia coli.

The molybdenum-containing nitrogenase contains an iron-molybdenum cofactor, whose synthesis involves at least six nif genes. Genes corresponding to nifE, N, B, and V occur in proximity in Clostridium pasteurianum, with nifN-B occurring as one gene and with nifV omega and nifV alpha in place of nifV. Between nifN-B and nifV omega V alpha, we found a gene whose sequence is similar to chlJ of Escherichia coli. chlJ is part of the chlD locus, which is involved in Mo transport. C. pasteurianum actively accumulates Mo in a process coregulated with nitrogen fixation. We propose that nifC is involved in Mo transport. The expression of nifC may be coregulated with nitrogen fixation because of the presence of nif-distinctive promoter and upstream sequences preceding nifC-nifV omega-nifV alpha. NifC contains a region typical of integral membrane proteins. Our findings suggest the involvement of a membrane-located nif gene product in Mo transport in C. pasteurianum.

Amino Acid Sequence↗

PubMed1976

Growth and nitrogen fixation of mutants defective in heterocyst differentiation in the blue-green alga Cylindrospermum sp.

Three classes of spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutants of the blue-green alga Cylindrospermum sp. were isolated in the presence of combined nitrogen. Mutants with complete loss of heterocyst differentiation formed longer filaments than the parent alga and were unable to grow in nitrogen-free medium and to fix nitrogen. The second type of mutants formed long filaments with terminal heterocysts. In the third type of mutants, paired proheterocyst differentiation occurred at regular intervals in long filaments and these were defective in their further development into mature heterocysts. Mutants with decreased heterocyst frequency due to defective differentiation and development showed less growth and nitrogen fixation in nitrogen-free medium than the parent alga.

Chloramphenicol↗

PubMed1996

Identification and sequence of a nifJ-like gene in Rhodospirillum rubrum: partial characterization of a mutant unaffected in nitrogen fixation.

A nifJ-like gene was identified in the photosynthetic purple non-sulphur bacterium Rhodospirillum rubrum. A DNA segment hybridizing to Klebsiella pneumoniae nifJ was isolated, the gene was inactivated, and a mutant strain, SNJ-1, was constructed by allele replacement. The mutation was confirmed by DNA sequencing. Northern blotting and by the lack of pyruvate oxidoreductase activity. This is the first report of a nifJ-like gene in photosynthetic bacteria. Unexpectedly, SNJ-1 was capable of nitrogen fixation, and growth was similar to the wild-type strain under all conditions investigated. Therefore, this is also the first demonstration that a nifJ homologue, when present, is not essential for nitrogen fixation in a diazotroph. The nifJ-like gene was sequenced and found to have considerable similarity to published nifJ gene sequences from other organisms. By primer extension, the initiation site for transcription was located, and a typical sigma 54 promoter sequence was identified.

Amino Acid Sequence↗

PubMed1997

The Klebsiella pneumoniae cytochrome bd' terminal oxidase complex and its role in microaerobic nitrogen fixation.

Cytochrome bd' has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy. In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported. The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex. The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium. Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products. It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity.

Aerobiosis↗

PubMed2002

Promiscuity of hosting nitrogen fixation in rice: an overview from the legume perspective.

The subject area of this review provides extraordinary challenges and opportunities. The challenges relate to the fact that the integration of various fields such as microbiology, biochemistry, plant physiology, eukaryotic as well as bacterial genetics, and applied plant sciences are required to assess the disposition of rice, an alien host, for establishing such a unique phenomenon as biological nitrogen fixation. The opportunities signify that, if successful, the breakthrough will have a significant impact on the global economy and will help improve the environment. This review highlights the literature related to the area of legume-rhizobia interactions, particularly those aspects whose understanding is of particular interest in the perspective of rice. This review also discusses the progress achieved so far in this area of rice research and the possibility of built-in nitrogen fixation in rice in the future. However, it is to be borne in mind that such research does not ensure any success at this point. It provides a unique opportunity to broaden our knowledge and understanding about many aspects of plant growth regulation in general.

Biotechnology↗

PubMed2026

Trade-off between photosynthetic promotion and nitrogen fixation suppression induced by chloroplast-targeted Mo nanoparticles in soybean.

Organelle-targeted nanomaterials offer opportunities to improve crop photosynthesis, yet their unintended effects on symbiotic nitrogen fixation remain poorly understood. Here, we developed chloroplast-targeted molybdenum nanoparticles (Chl-Mo) and compared their effects with those of ionic Mo (IonMo) and non-targeted Mo nanoparticles in soybean. Chl-Mo preferentially accumulated in chloroplasts, enhancing photosynthetic carbon assimilation, thylakoid development, PSII performance, sucrose transport, and biomass accumulation. However, this growth promotion was accompanied by suppressed nodule nitrogenase activity, reduced nif gene expression, inhibited GS/GOGAT-mediated nitrogen assimilation, and disrupted microoxic and ROS homeostasis in nodules. Integrated nodule proteomics and metabolomics showed downregulation of sucrose transport, glycolysis, pyruvate metabolism, and amino acid biosynthesis, indicating a decoupling between enhanced carbon input and nitrogen utilization. Root transcriptomics further revealed oxidative stress, impaired nitrate assimilation, and attenuated early symbiotic signaling. These findings demonstrate that chloroplast-targeted Mo delivery can enhance photosynthesis while compromising symbiotic nitrogen fixation, highlighting the need to evaluate belowground symbiotic functions when developing organelle-targeted nanotechnologies for sustainable agriculture.

Chloroplast-targeted Mo↗

PubMed2000

Evolution of nitrogen fixation in spatially structured populations of Rhizobium.

Symbiosis between legumes and nitrogen-fixing bacteria is thought to bring mutual benefit to each participant. However, it is not known how rhizobia benefit from nodulation of legume hosts because they fix nitrogen only after differentiating into bacteroids, terminally differentiated cells that cannot reproduce. Because free-living rhizobia can reproduce, and may benefit from the increase of plant root exudates stimulated by nodulation, evolution of symbiotic nitrogen fixation may depend upon kin selection. However, unrelated nonmutualists may also benefit from increased plant exudates and nitrogen-fixing populations are therefore vulnerable to invasion by nonfixing, saprophytic Rhizobium. The access of nonfixing Rhizobium to the plant exudates associated with nodules depends upon the spatial structure of the Rhizobium populations within the soil. We investigate the influence of spatial structure on the evolution of N-fixation within a Rhizobium population using a mathematical model. Our model demonstrates that spatial structure is necessary for the evolution of N-fixation and that N-fixation is more likely to evolve with increasing degrees of spatial structure. In fact, we identify three dynamic outcomes that depend upon the relative strength of the costs of N-fixation relative to the degree of spatial structure and benefits resulting from nodulations. If the costs are relatively high, N-fixation will not evolve; if the costs are relatively low, N-fixing genes will fix in the population, but at intermediate conditions, a stable mixture of N-fixing bacteria and nonfixing bacteria will be maintained. The conditions for coexistence of N-fixing bacteria and nonfixing bacteria expand under a saturating relationship between nodule numbers and N-fixing genotype frequency.

Biological Evolution↗

PubMed1994

ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.

Alleles↗

PubMed1991

Nitrogen Fixation in Peanut Nodules during Dark Periods and Detopped Conditions with Special Reference to Lipid Bodies.

The peanut plant (Arachis hypogaea L.), unlike other known legumes, can sustain nitrogen fixation when prolonged periods of darkness or detopping curtail the supply of photosynthate to the nodule. This ability to withstand photosynthate stress is attributed to the presence of lipid bodies in infected nodule cells. In both dark-treated and detopped plants, the lipid bodies show a gradual decrease in numbers, suggesting their utilization as a source of energy and carbon for nitrogen fixation. Lipolytic activity can be localized in the lipid bodies, and the existence of beta-oxidation pathway and, glyoxylate cycle is shown by the release of (14)CO(2) from (14)C lineoleoyl coenzyme A by the nodule homogenate.

Journal Article↗