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PubMed1979

Environmental and genotypic effects on the respiration associated with symbiotic nitrogen fixation in peas.

Estimated values for the respiration associated with symbiotic nitrogen fixation in Pisum sativum L. were independent of irradiance, temperature, plant age, and CO(2) concentration, despite large variation in the total rates of C(2)H(2) reduction and root + nodule respiration. Similar values were also found in Phaseolus vulgaris L., Vicia faba L. and Glycine max (L.) Merr. Among all combinations of four Pisum cultivars with four Rhizobium leguminosarum inoculants only the plant genotype significantly affected the fixation-linked respiration, although both plant and bacterial types significantly influenced the total rate of C(2)H(2) reduction. On the basis of measured rates of H(2) evolution and C(2)H(2) reduction, or total nitrogen gain in the same system, the least respiration per unit of ammonia produced symbiotically was estimated as 4.8 to 6.9 moles CO(2) (mole NH(3))(-1) in Laxton's Progress and the greatest as 9.3 to 13.3 moles CO(2) (mole NH(3))(-1) in an Indian cultivar, as compared to a theoretical minimum respiration requirement of 4.7 moles CO(2) (mole NH(3))(-1) in peas.

Journal Article↗
PubMed1993

In vitro activity of NifL, a signal transduction protein for biological nitrogen fixation.

In the free-living diazotroph Klebsiella pneumoniae, the NifA protein is required for transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself. NifA activates transcription of nif operons by the alternative holoenzyme form of RNA polymerase, sigma 54 holoenzyme. In vivo, NifL is known to antagonize the action of NifA in the presence of molecular oxygen or combined nitrogen. We now demonstrate inhibition by NifL in vitro in both a coupled transcription-translation system and a purified transcription system. Crude cell extracts containing NifL inhibit NifA activity in the coupled system, as does NifL that has been solubilized with urea and allowed to refold. Inhibition is specific to NifA in that it does not affect activation by NtrC, a transcriptional activator homologous to NifA, or transcription by sigma 70 holoenzyme. Renatured NifL also inhibits transcriptional activation by a maltose-binding protein fusion to NifA in a purified transcription system, indicating that no protein factor other than NifL is required. Since inhibition in the purified system persists anaerobically, our NifL preparation does not sense molecular oxygen directly.

ATP-Binding Cassette Transporters↗
PubMed1985

Lesions in citrate synthase that affect aerobic nitrogen fixation by Azotobacter chroococcum.

A class of Azotobacter chroococcum mutants induced by Tn1 that were defective in normal aerobic nitrogen fixation when grown on sugars (Fos-) were corrected by provision of alpha-ketoglutarate or glutamate. In a representative mutant, Fos252, rates of evolution of 14CO2 from [14C]acetate or [14C]glucose were 5% of the parental values, although uptake and incorporation were normal for both substrates. The results suggest that a lesion affects the entry of substrates into the tricarboxylic acid cycle. The activity of citrate synthase in Fos252 in vitro was 5% that of the parents. The citrate synthase (gltA) gene from Escherichia coli was cloned into broad-host-range vectors and mobilized into Fos252. The plasmids restored parental citrate synthase activities to Fos252 and complemented the inability to fix N2 in air. The data indicate that a mutation causing an intrinsic limitation in respiratory capacity abolishes normal aerobic N2 fixation, which is consistent with the hypothesis of respiratory protection for nitrogenase in Azotobacter species.

Azotobacter↗
PubMed2001

Measurement of symbiotic nitrogen-fixation in leguminous host-plants grown in heavy metal-contaminated soils amended with sewage sludge.

Rates of nitrogen fixation by Rhizobium in symbiosis with leguminous host-plants including white clover, broad bean and peas have been established in soils that have been amended experimentally with heavy metal-contaminated sewage sludges. Results from 15N-dilution experiments for the measurement of N2 fixation have shown that adverse heavy metal effects are apparent on symbiotic N2 fixation rates for white clover grown in inter-specific competition with ryegrass under mixed sward conditions, compared to white clover grown in pure sward. Further experiments on broad bean and pea indicated a significant, but minor-inhibitory metal-related effect on the rate of N2 fixation compared to untreated soils and soils amended with a relatively uncontaminated sludge. The implications of the results with respect to sludge utilisation in agriculture are discussed.

Agriculture↗
PubMed2002

The H-NS-like protein HvrA modulates expression of nitrogen fixation genes in the phototrophic purple bacterium Rhodobacter capsulatus by binding to selected nif promoters.

Genetic analyses based on chromosomal lac fusions to nitrogen fixation (nif) genes demonstrated that NifA-dependent transcriptional activation of expression of Rhodobacter capsulatus nifH and nifB1 was negatively modulated by HvrA, whereas regulation of rpoN, nifA1, and nifA2 was independent of HvrA. Expression of hvrA itself was not influenced by a mutation in ntrC, which is absolutely essential for N(2) fixation. Furthermore, HvrA accumulated to comparable levels in the presence and absence of ammonium, suggesting that the amount of HvrA in the cells does not differ under nitrogenase-repressing or -derepressing conditions. In addition, competitive gel retardation studies with HvrA-His(6) purified from R. capsulatus were carried out, demonstrating preferential binding of HvrA to the nifH promoter region.

Bacterial Proteins↗
PubMed2006

Phosphoenolpyruvate carboxylase plays a crucial role in limiting nitrogen fixation in Lotus japonicus nodules.

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is believed to play a significant role in supporting nitrogen fixation via anaplerotic CO2 fixation for recycling carbon in nodules. Using the antisense technique, we decreased the expression levels of the nodule-enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) in order to look at the influence of the symbiotic phenotype and biochemical parameters. Three independent transgenic L. japonicus plants (designated as Asppc1, Asppc2 and Asppc3) were prepared using a Ljpepc1 DNA fragment which is under the control of the cauliflower mosaic virus 35S promoter. Extensive suppression of the Ljpepc1 transcript in nodules of Asppc plants (T3 homologous plants) was confirmed by RNA gel blot, Western blot and enzyme activity assays. In nodules of Asppc plants, PEPC activity was reduced to about 10% of that of non-transformants and the plants showed typical nitrogen-deficient symptoms without a supply of nitrogen nutrient, and returned to normal growth when nitrate was supplied at 2.5 mM. The acetylene reduction activity per fresh weight of nodules of these Asppc plants decreased by 29% at 35 dai (days after infection). Various enzyme activities and metabolite levels were surveyed using Asppc plants at 35 dai. Significant reduction of sucrose synthase and asparagine aminotransferase activities was observed in Asppc nodules. In addition, sucrose, succinate, asparagine, aspartate and glutamate contents also decreased in Asppc nodules. The data are discussed in terms of a role for PEPC in the carbon/nitrogen metabolic flux in nodules.

Acetylene↗
PubMed2004

Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus.

Research on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic pathways in many different legume species. We utilized the tools of transcriptomics and metabolomics to obtain an unprecedented overview of the metabolic differentiation that results from nodule development in the model legume, Lotus japonicus. Using an array of more than 5000 nodule cDNA clones, representing 2500 different genes, we identified approximately 860 genes that were more highly expressed in nodules than in roots. One-third of these are involved in metabolism and transport, and over 100 encode proteins that are likely to be involved in signalling, or regulation of gene expression at the transcriptional or post-transcriptional level. Several metabolic pathways appeared to be co-ordinately upregulated in nodules, including glycolysis, CO(2) fixation, amino acid biosynthesis, and purine, haem, and redox metabolism. Insight into the physiological conditions that prevail within nodules was obtained from specific sets of induced genes. In addition to the expected signs of hypoxia, numerous indications were obtained that nodule cells also experience P-limitation and osmotic stress. Several potential regulators of these stress responses were identified. Metabolite profiling by gas chromatography coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis.

Expressed Sequence Tags↗
PubMed1982

Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae.

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 mug or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his strain, and was also Nif on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.

Journal Article↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1976

The effect of rhizobiophages on the effectiveness of Rhizobium meliloti in symbiosis with lucerne. I. Effect of various strains of R. meliloti and rhizobiophages on the yield and nitrogen fixation in lucerne.

Pot and test-tube experiments were carried out on the effect of phages on yield and nitrogen fixation in lucerne inoculated with effective and non-effective strains of Rhizobium meliloti. It was found that some strains of R. meliloti became activated by the phages, which was reflected in an increase in yield and in crude protein content in the plants, while others lost their effectiveness. The results of pot experiments were in line with those of the test-tube experiments and have demonstrated that the effect of phages on the effectiveness of Rhizobium is not simply negative or positive, but depends on the properties of the bacterial strains used.

Bacteriophages↗
PubMed2001

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413.

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.

Amino Acid Sequence↗
PubMed1982

Screening and selection of maize to enhance associative bacterial nitrogen fixation.

The ability of maize (corn, Zea mays L.) to support bacterial nitrogen fixation in or on maize roots has been increased, through screening and selection. Isotopic N fixed from (15)N(2) was found on the roots. The nitrogen-fixing association was found in germplasm from tropical maize, but this activity can be transferred to maize currently used in midwestern United States agriculture.

Journal Article↗
PubMed1989

Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon.

A second nitrogen fixation (nif) operon in the cyanobacterium (blue-green alga) Anabaena (Nostoc) sp. strain PCC 7120 has been identified and sequenced. It is located just upstream of the nifHDK operon and consists of four genes in the order nifB, fdxN, nifS, and nifU. The three nif genes were identified on the basis of their similarity with the corresponding genes from other diazotrophs. The fourth gene, fdxN, codes for a bacterial type ferredoxin (Mulligan, M. E., Buikema, W. J., and Haselkorn, R. (1988) J. Bacteriol. 167, 4406-4410). The four genes are probably transcribed as a single operon, but are expressed at a lower level than the nifHDK operon, and only after a developmentally induced DNA rearrangement occurs that excises a 55-kilobase pair element from within the fdxN gene (Golden, J. W., Mulligan, M. E., and Haselkorn, R. (1987) Nature 327, 526-529; Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J., and Haselkorn, R. (1988) J. Bacteriol. 170, 5034-5041). The promoter for the nifB operon was located by primer extension. Comparison of the nifB 5'-flanking sequence with the nifH 5'-flanking sequence did not reveal any consensus base pairs that would define a nif promoter for Anabaena. The operon contains two instances of 7-base pair directly repeated sequences: seven copies of the repeated sequence are found between the nifB and fdxN genes and six copies are found between the nifS and nifU genes. The function of these repeats is unknown.

Amino Acid Sequence↗
PubMed1983

Growth and nitrogen fixation by cultures of Bacillus sp. S 77 g, Pseudomonas sp. S 44 b, and Klebsiella sp. S 145 m, isolated from Egyptian soils.

Study of the behaviour of representative aerobic freeliving dinitrogen-fixers, other than azotobacters, with respect to different carbon sources, showed that a broad range of compounds is accessible to them. This group of nitrogen-fixers can assimilate molecular nitrogen as a result of utilization of carbohydrates, alcohols, and organic acid salts. The highest nitrogen-fixation productivity, reaching 20.6-27.3 mg of nitrogen per g of carbon utilized, was observed on medium containing glucose as the carbon and energy source. Supplementary growth factors added to the N-deficient medium in the form of small amounts of yeast extract stimulated their growth and fixation of atmospheric nitrogen. This stimulation is particularly evident in mixed cultures. The nature of the mutual influences of micro-organisms in these cultures remains fairly obscure and further inquiry is necessary to elucidate this problem. A medium containing 1% glucose and Difco yeast extract to produce a bound nitrogen concentration of 10 ppm appeared to be the most favourable for the cultivation and N2-fixation of this group of microorganisms.

Bacillus↗
PubMed2001

Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii.

Several solvent-producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of 15N2 into cellular material. The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVomega and nifValpha genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifVomega genes of C. pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase.

Acetylene↗
PubMed1998

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum.

A spontaneous mutant derivative of Azotobacter vinelandii CA12 (delta nif HDK), which vanadium-dependent nitrogen fixation is not inhibited by molybdenum (A. vinelandii CARR), grows profusely on BNF-agar containing 1 microM Na2MoO4, alone or supplemented with 1 microM V2O5. The expression of A. vinelandii vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CARR was not inhibited by 1 mM Na2MoO4, whereas molybdenum at much lower concentration inhibited the expression of vnfH::lacZ and vnfA::lacZ fusions in A. vinlandii CA12. The mutant also exhibited normal acetylene reduction activity in the presence of 1 microM Na2MoO4. The expression of A. vinelandii nifH::lacZ fusion in A. vinelandii CARR was low even though the cells were cultured under non-repressing conditions with urea as nitrogen source in the presence of Na2MoO4. The molybdenum content of A. vinelandii CARR cells was found to be about one-fourth that of A. vinelandii CA12. No nitrate reductase activity could be detected in A. vinelandii CARR when the cells were cultured in the presence of 10 microM Na2MoO4, whereas A. vinelandii CA12 exhibited some activity even with 100 pM Na2MoO4.

Acetylene↗
PubMed1982

Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.

Bacterial Proteins↗