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PubMed1993

Genes at different regulatory levels are required for the ammonia control of nodulation in Rhizobium meliloti.

The expression of the nodulation genes nodABC of Rhizobium meliloti, which determine early response functions to plant host signals, is regulated by the level of ammonia, the primary product of symbiotic nitrogen fixation. We show that the pathway that links the ammonia-induced signal to the transcriptional control of the nodABC genes involves at least two regulatory levels. The fluctuating nitrogen level is sensed and the signal is mediated by the members of the general nitrogen regulatory (ntr) system, then transmitted to the syrM-nodD3 genes representing the nod-specific level of ammonia regulation. At low ammonia concentration, the activator protein NtrC exerts its effect via nodD3. In conditions of nitrogen excess ntrR, involved in the repression of nod genes, may function in coordination with the syrM gene. Finally, the NodD3 protein may relay the nitrogen status signal to the transcriptional control of the nodABC genes.

Ammonia↗
PubMed2000

Internal glutamine and glutamate pools in Klebsiella pneumoniae grown under different conditions of nitrogen availability.

Internal pool sizes of glutamine and glutamate in Klebsiella pneumoniae grown under nitrogen limitation or nitrogen sufficiency were measured to study the signal transduction of external nitrogen limitation. K. pneumoniae cells were grown in an anaerobic, ammonium-limited chemostat culture. At a growth rate of 0.217 h(-1), the steady state ammonium concentration in the culture was 55 microm, correlating with repression of the nitrogen fixation (nif) genes. At growth rates below 0.138 h(-1), the ammonium concentration in the culture dropped below 0.5 microm and the nif genes became derepressed. During the transition from nitrogen sufficiency to nitrogen limitation, the internal glutamine pool in K. pneumoniae decreased by a factor of approximately 6. The glutamate pool, however, remained stable. Similarly, in anaerobic batch cultures with different limiting nitrogen sources, the glutamine pool generally decreased by a factor of 7 to 9 when nif gene derepression was achieved. All the limiting nitrogen sources used resulted in decreased growth rates compared with growth under nitrogen excess, suggesting an inverse relationship between glutamine pool size and doubling time. These studies indicate that K. pneumoniae perceives external nitrogen limitation as internal glutamine limitation.

Fermentation↗
PubMed1982

Rhizobium meliloti mutants altered in ammonium utilization.

Derivatives of Rhizobium meliloti 2011 required trace amounts of glutamate to use ammonium as the nitrogen source for growth, although they could use serine as the sole nitrogen source. Specific activities of ammonium assimilatory enzymes were similar to those in strain Rm2011. The mutants were deficient in nitrogen fixation.

Ammonium Chloride↗
PubMed1976

Characterization of a nitrogen-fixing bacterial strain from the roots of Digitaria sanguinalis.

Rates of nitrogen fixation of 3 to 10 g of N2 fixed per hectare per day were associated with root systems of Digitaria sanguinalis. A Gram-negative motile aerobic bacterial strain that was capable of N2 fixation was isolated from a washed root sample of one of these plants. Optimal growth and N2 fixation occurred at a pH of about 6.5, a temperature of 30-37 degrees C, and at a pO2 of about 0.01 atm. Increased rates of N2 fixation resulted when this strain was grown in mixed cultures with aerobic or facultative bacteria. Observations of cellular and cultural morphology and results of biochemical and physiological studies indicate that the isolate may be related to the Azotobacteraceae but that it is not identical with any of the members of this family. The importance of N2 fixation by this isolate in nature is unknown.

Acetylene↗
PubMed2000

[Mycorrhizal research on nitrogen-fixing plants].

Advances in mycorrhizal research on nitrogen-fixing plants were introduced. The universality of mycorrhizal formation in nitrogen fixing plants, the reciprocal promotion effect of symbiosis, and the application prospect of mycorrhizal techniques under stressed conditions were summarized and reviewed. The mechanism of enhancing nitrogen fixation of N-fixing plants by mycorrhizal fungi in symbiosis was preliminary discussed.

Fungi↗
PubMed1975

The history of inorganic nitrogen in the biosphere.

When in the primeval atmosphere ammonia approached exhaustion, bacteria resembling clostridia developed mechanisms for nitrogen fixation. The fixation was continued by the photosynthetic bacteria. In the later, oxidizing, atmosphere the combined activities of the nitrificants and the denitrificants could lead to a large-scale cyclic regeneration of free nitrogen. The possibility of a descent of the nitrificants from hypothetical photosynthetic bacteria, which used ammonia as electron donor, is discussed. The anoxygenic atmosphere contained no nitrate, and therefore neither nitrate fermentation nor nitrate respiration were precursors of aerobic respiration. This evolved from photosynthesis. In nitrate fermentation, nitrate serves only as an incidental electron acceptor; this process is merely an evolutionary sideline. Nitrate respiration evolved from aerobic respiration. While in present conditions the reaction of nitrogen with oxygen and water to give nitrate is exergonic and possibly occurs at a low rate, the antagonistic action of the denitrificants maintains the stationary concentrations of nitrogen and oxygen in the air.

Aerobiosis↗
PubMed2002

Performance of an age series of Alnus-cardamom plantations in the Sikkim Himalaya: nutrient dynamics.

Nutrient cycling, nutrient use efficiency and nitrogen fixation in an age series of Alnus-cardamom plantations were studied in the eastern Himalaya. The impact of stand age (5, 10, 15, 20, 30 and 40 years) on the nutrient dynamics of mixtures of N2-fixing (Alnus nepalensis) and non-N2-fixing (large cardamom) plants was assessed. Foliar nutrient concentrations of Alnus decreased with advancing age groups of plantations and showed an inverse relationship with stand age. Annual N fixation increased from the 5-year-old stand (52 kg ha(-1)), peaking in the 15-year-old stand (155 kg ha(-1)) and then decreased with increasing plantation age. Nitrogen and phosphorus uptake was lowest in the 40-year-old stand, and highest in the 15- and 5-year-old stand, respectively. Nutrient storage in understorey cardamom was very high: up to 31 % N and 59 % P of the stand total in the 15-year-old stand. Nutrient use efficiency was higher (with faster turnover times) in younger stands and decreased (with slower turnover times) in older plantations. Nitrogen retranslocation showed a strong positive relationship with stand age, while that of P was inversely related to stand age. Nutrient standing stock, uptake and return were also highest in the 15-year-old stand. Nitrogen and P cycling in Alnus-cardamom plantations was functionally balanced. Nutrient cycling and dynamics indicated that Alnus-cardamom plantations performed sustainably up to 15-20 years. The management practice should be altered to incorporate replantation after this age.

Algorithms↗
PubMed2006

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress.

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.

Adaptation, Physiological↗
PubMed1983

Transfer and expression of Klebsiella nif genes in Alcaligenes faecalis, a nitrogen-fixing bacterium associated with rice root.

Plasmid pRD1 carrying Klebsiella nif genes was found to be transferable by conjugation from Escherichia coli JC5466 (pRD1) to Alcaligenes faecalis A-15 at a frequency of 5 X 10(-4). Nitrogenase activity of four A-15 (pRD1) strains tested was found to be higher than that of their parent A-15, as determined by the acetylene reduction assay. A-15-1 was a Nif- mutant derived from A-15. After mating with JC5466 (pRD1), the nitrogenase activity was restored. PRD1 was stable in A. faecalis and could be transferred to E. coli JC5466-1 by conjugation. The fact that Klebsiella nif genes carried in pRD1 can be expressed in A. faecalis makes it possible to use pRD1 as a tool for genetic analysis and genetic engineering of the nitrogen fixation system in A. faecalis.

Alcaligenes↗
PubMed2003

The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions.

The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting.

Azotobacter vinelandii↗
PubMed1988

Identification of a new Bradyrhizobium japonicum gene (frxA) encoding a ferredoxinlike protein.

An open reading frame of 74 codons was identified downstream of the nifB gene of Bradyrhizobium japonicum 110. The predicted amino acid sequence shared 63% similarity with the Rhodopseudomonas palustris ferredoxin I sequence. We propose to name the gene frxA. The frxA gene was found to be cotranscribed with the nifB gene. An insertion mutation within frxA hardly affected nitrogen fixation activity.

Amino Acid Sequence↗
PubMed1994

Cadmium mediated control of nitrogenase activity and other enzymes in a nitrogen fixing cyanobacterium.

Cadmium decreased the specific growth rate and total cell mass at concentrations > 0.001 micrograms ml-1. It also reduced heterocyst frequency, total cell protein, nitrogenase activity (acetylene reduction) and decreased glutamine synthetase (GS) and glutamate synthase (GOGAT) activities. However, cells grown under low concentration of cadmium (0.001 microgram ml-1) had enhanced nitrogenase as well as GS activity besides showing increased growth rate and heterocyst frequency. Cadmium has apparently controlled nitrogenase activity and nitrogen fixation possibly via mediating enzymes including glutamine synthetase in Nostoc muscorum.

Cadmium↗
PubMed2004

Intracellular spheroid bodies of Rhopalodia gibba have nitrogen-fixing apparatus of cyanobacterial origin.

Nitrogen fixation is not regarded as a eukaryotic invention. The process has only been reported as being carried out by bacteria. These prokaryotes typically interact with their eukaryotic hosts as extracellular and temporary nonobligate nitrogen-fixing symbionts. However, intracellular permanent "spheroid bodies" have been reported within the fresh-water diatom Rhopalodia gibba, and these, too, have been speculated as being able to provide nitrogen to their host diatom. These spheroid bodies have gram-negative characteristics with thylakoids. We demonstrate that they fix nitrogen under light conditions. We also show that phylogenetic analyses of their 16rRNA and nif D genes predict that their genome is closely related to that of Cyanothece sp. ATCC 51.142, a free-living diazotrophic cyanobacterium. We suggest that the intracellular spheroid bodies of Rhopalodia gibba may represent a vertically transmitted, permanent endosymbiotic stage in the transition from a free-living diazotrophic cyanobacterium to a nitrogen-fixing eukaryotic organelle.

Algal Proteins↗
PubMed1993

Sequence, genetic, and lacZ fusion analyses of a nifR3-ntrB-ntrC operon in Rhodobacter capsulatus.

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are glnB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus. nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli regulatory gene, fis. Analysis of ntrC-lacZ fusions and complementation data indicate that nifR3 ntrBC constitute a single operon. nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription). This gene organization suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R. capsulatus.

Amino Acid Sequence↗
PubMed1988

Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid.

Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.

Cloning, Molecular↗
PubMed1996

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae.

In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription by sigma54-holoenzyme. In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen. In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 microM) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogen-limiting conditions. Adding back Fe in only twofold molar excess over phenanthroline restores NifA activity, whereas adding other metals fails to do so. By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline. Hence, Fe is required to relieve the inhibition of NifA activity by NifL in vivo. Despite the Fe requirement in vivo, we have found no evidence that NifL contains Fe or an iron-sulfur (Fe-S) cluster. Determination of the molecular mass of an inhibitory form of NifL overproduced under aerobic conditions indicated that it was not posttranslationally modified. When NifL was synthesized in vitro, it inhibited transcriptional activation by NifA even when it was synthesized under anaerobic conditions in the presence of a high Fe concentration or of superoxide dismutase, which is known to protect some Fe-S clusters. Moreover, overproduction of superoxide dismutase in vivo did not relieve NifL, inhibition under aerobic conditions, and attempts to relieve NifL inhibition in vitro by reconstituting Fe-S clusters with the NifS enzyme (Azotobacter vinelandii) were unsuccessful. Since we obtained no evidence that Fe acts directly on NifL or NifA, we postulate that an additional Fe-containing protein, not yet identified, may be required to relieve NifL inhibition under anaerobic, nitrogen-limiting conditions.

Aerobiosis↗
PubMed2002

Regulatory response of Methanococcus maripaludis to alanine, an intermediate nitrogen source.

In the methanogenic archaeon Methanococcus maripaludis, growth with ammonia results in conditions of nitrogen excess. Complete repression of nitrogen fixation (nif) gene transcription occurs, and glutamine synthetase (glnA) gene transcription falls to a basal constitutive level. In addition, ammonia completely switches off nitrogenase enzyme activity. In contrast, growth with dinitrogen as the sole nitrogen source results in nitrogen starvation, full expression of nif and glnA, and high activity of nitrogenase. Here we report that a third nitrogen source, alanine, results in an intermediate regulatory response. Growth with alanine resulted in intermediate transcription of nif and glnA, and addition of alanine to a nitrogen-fixing (diazotrophic) culture caused partial switch-off of nitrogenase. This uniformity of response occurred despite differences in regulatory mechanisms. Nitrogenase switch-off requires the nitrogen sensor homologs NifI(1) and NifI(2), while transcriptional regulation of nif and glnA relies on a different, unknown sensor mechanism. In addition, although nif and glnA transcription are governed by a common repressor, the numbers and arrangements of repressor binding sites differ. Thus, the nif promoter region contains two operators situated downstream of the transcription start site, while the glnA promoter region contains only one operator just upstream of two closely spaced transcription start sites. In a previous study of nif expression using ammonia, we were able to detect a role only for the first nif operator in repression. Here we show that nif repression by alanine requires the second operator as well. In contrast, in the case of glnA the single operator was sufficient for repression by ammonia or alanine. These results suggest a uniform cellular response to nitrogen that is mediated by a different mechanism in each case.

Alanine↗