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PubMed2005

Endophytic colonization and in planta nitrogen fixation by a diazotrophic Serratia sp. in rice.

Nitrogen fixing endophytic Serratia sp. was isolated from rice and characterized. Re-colonization ability of Serratia sp. in the rice seedlings as endophyte was studied under laboratory condition. For detecting the re-colonization potential in the rice seedlings, Serratia sp. was marked with reporter genes (egfp and Kmr) using transposon mutagenesis. The conjugants were screened for re-colonization ability and presence of nif genes using PCR. Further, the influence of flavonoids and growth hormones on the endophytic colonization and in planta nitrogen fixation of Serratia was also investigated. The flavonoids, quercetin (3 microg/ml) and diadzein (2 microg/ml) significantly increased the re-colonization ability of the endophytic Serratia, whereas the growth hormones like IAA and NAA (5 microg/ml) reduced the endophytic colonization ability of Serratia sp. Similarly, the in planta nitrogen fixation by Serratia sp. in rice was significantly increased due to flavonoids. The inoculation of endophytic diazotrophs increased the plant biomass and biochemical constituents.

Biomass↗

PubMed1990

Measurement of nitrogen fixation by soybean in the field using the ureide and natural N abundance methods.

Nitrogen fixation by field-grown soybean (Glycine max [L.] Merrill) was assessed by the natural (15)N abundance and ureide methods. The field sites (five) and genotypes (six, plus two levels of inoculation on Bragg) were chosen to provide a range of proportions of plant N derived from nitrogen fixation (P). Genotypes K466, K468, nts1007, and nts1116 and Davis were included on the basis of their reported tolerance of the suppressive effects of nitrate on nodulation and nitrogen fixation. Bragg was included as a ;nitrate-sensitive' genotype. Seeds of all genotypes were inoculated at sowing with Bradyrhizobium japonicum CB1809 (USDA136). Amounts of nitrate in the soil profile (0-1.2 meter depth) at sowing ranged from 70 (site 3) to 278 kilograms per hectare (site 5), resulting in large effects on plant nodulation, on the delta(15)N values of nodulated plants, on the relative abundance of ureide-N in vacuum-extracted sap (VES) and stem extracts, and finally on the estimates of P. There was no relationship between amount of soil nitrate at sowing and the delta(15)N of the plant-available soil N. Correlation matrices of the measured and calculated parameters indicated generally weak correlations between crop growth (dry matter and N) and the parameters of symbiotic activity (nodule weight, delta(15)N, relative ureide-N); correlations were strong and highly significant between nodulation and the measures of nitrogen fixation (delta(15)N, relative ureide-N; r = 0.79-0.92). Estimates of P ranged between 0 and 68% (delta(15)N) and between 6 and 56% (ureide) and were highly correlated (r = 0.97). Results indicated that the ureide method can be used with confidence to assess P by field-grown crops of soybean.

Journal Article↗

PubMed2004

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii.

NifL is an antiactivator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA, a member of the family of sigma(54)-dependent transcriptional activators. Although the C-terminal region of A. vinelandii NifL shows homology to the transmitter domains of histidine protein kinases, signal transduction between NifL and NifA is conveyed by means of protein-protein interactions rather than by phosphorylation. Binding of the ligand 2-oxoglutarate to NifA plays a crucial role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. We have used a suppressor screen to identify a critical arginine residue (R306) in NifL that is required to release NifA from inhibition under appropriate environmental conditions. Amino acid substitutions at position 306 result in constitutive inhibition of NifA activity by NifL, thus preventing nitrogen fixation. Biochemical studies with one of the mutant proteins demonstrate that the substitution alters the conformation of NifL significantly and prevents the response of NifA to 2-oxoglutarate. We propose that arginine 306 is critical for the propagation of signals perceived by A. vinelandii NifL in response to the redox and fixed-nitrogen status and is required for a conformational switch that inactivates the inhibitory function of NifL under conditions appropriate for nitrogen fixation.

Adenosine Diphosphate↗

PubMed1988

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed2003

Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis.

The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume-rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.

Amino Acids↗

PubMed1999

Culture-independent characterization of a gene responsible for nitrogen fixation in the symbiotic microbial community in the gut of the termite Neotermes koshunensis.

Expression of the nitrogen fixation gene, nifH, in the gut of the termite Neotermes koshunensis was characterized without cultivation. nifH cDNA was directly amplified from mRNA of the mixed microbial population in the gut by reverse transcription (RT)-PCR. Analyses of the RT-PCR products revealed that, among the diverse nifH sequences, only a few corresponding to an alternative nitrogenase (encoded by the anf gene) were preferentially transcribed in the termite gut. Expression of the anf gene was further investigated quantitatively under several termite feeding conditions by competitive PCR. The levels of expression of the anf gene were largely congruent with the nitrogen fixation activity displayed by the termite. The amounts of the genomic anf gene in the population showed no significant change, indicating that the level of expression was critical for nitrogen fixation activity. Interestingly, no significant decrease in the expression level was observed when the diet contained molybdenum (Mo), which represses ordinary anf genes. A 3.6-kb DNA region downstream of the anf gene was isolated and found to contain reading frames homologous to anfH, anfD, and anfG of the Bacteria domain which encode subunits of an alternative nitrogenase having no Mo as a cofactor. This DNA region also contained reading frames encoding glnB-like proteins, which is a common feature of the nitrogenase genes of the Archaea domain. These results indicate that the anf group of nitrogenase genes is the most important group of genes responsible for nitrogen fixation in N. koshunensis and that the anf gene possesses novel features with respect to the regulation of its expression and its gene organization.

Animals↗

PubMed1987

Molecular aspects of nitrogen fixation by photosynthetic prokaryotes.

The photosynthetic prokaryotes possess diverse metabolic capabilities, both in carrying out different types of photosynthesis and in their other growth modes. The nature of the coupling of these energy-generating processes with the basic metabolic demands of the cell, such as nitrogen fixation, has stimulated research for many years. In addition, nitrogen fixation by photosynthetic prokaryotes exhibits several unique features; the oxygen-evolving cyanobacteria have developed various strategies for protection of the oxygen-labile nitrogenase proteins, and some photosynthetic bacteria have been found to regulate their nitrogenase (N2ase) activity in a rapid response to fixed nitrogen, thus saving substantial amounts of energy. Recent advances in the biochemistry, physiology, and genetics of nitrogen fixation by cyanobacteria and photosynthetic bacteria are reviewed, with special emphasis on the unique features found in these organisms. Several major topics in cyanobacterial nitrogen fixation are reviewed. The isolation and characterization of N2ase and the isolation and sequence of N2ase structural genes have shown a great deal of similarity with other organisms. The possible pathways of electron flow to N2ase, the mechanisms of oxygen protection, and the control of nif expression and heterocyst differentiation will be discussed. Several recent advances in the physiology and biochemistry of nitrogen fixation by the photosynthetic bacteria are reviewed. Photosynthetic bacteria have been found to fix nitrogen microaerobically in darkness. The regulation of nif expression and possible pathways of electron flow to N2ase are discussed. The isolation of N2ase proteins, particularly the covalent modification of the Fe protein, the nature of the modifying group, properties of the activating enzyme, and regulating factors of the inactivation/activation process are reviewed.

Aerobiosis↗

PubMed1990

Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods.

We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max [L.] Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using (15)N dilution. In one experiment with CB1809, plants were supplied throughout growth with either N-free nutrients or with nutrients supplemented with 1, 2, 4, or 8 millimolar (15)N-nitrate and harvested on eight occasions between V6 and R7 for root-bleeding sap, vacuum-extracted sap, stems (including petioles), and whole plant dry matter. Analyses of the saps and stem extracts for ureides (allantoin plus allantoic acid), alpha-amino-N, and nitrate, and of dry matter for N and (15)N, indicated a positive effect of nitrate supply on concentrations of nitrate in saps and extracts and a negative effect on ureides and on the proportion of plant N derived from nitrogen fixation. The relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap (100 [ureide-N]/[ureide-N+ alpha-amino-N + nitrate-N]) and stem extracts (100 [ureide-N]/[ureide-N + nitrate-N]) and the proportion of plant N, derived from nitrogen fixation between successive samplings were highly correlated (r = 0.97-1.00). For each variable, two standard curves were prepared to account for the shifts in the compositions of N solutes of xylem saps and extracts after flowering which were not related to a change in nitrogen fixation. Relationships between relative ureide-N and the proportion of plant N derived from nitrogen fixation were not affected by plant genotype or by strain of rhizobia. Therefore, assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules.

Journal Article↗

PubMed1982

Nitrogen fixation in Klebsiella pneumoniae during osmotic stress. Effect of exogenous proline or a proline overproducing plasmid.

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity of Klebsiella pneumoniae, wild-type strain M5A1. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F' conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixation by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.

Klebsiella pneumoniae↗

PubMedDate not supplied

Intensity of nitrogen fixation in yellow lupine in presence of different doses of mineral nitrogen.

The effect of different doses of mineral nitrogen on the nitrogen-fixing activity of lupine nodules during vegetation was studied. It was shown that 0.5 of the dose of mineral nitrogen had an inhibitory effect only in the initial period of development of the nodules, whereas full and double doses of mineral nitrogen inhibited nitrogen fixation throughout the period of vegetation. Supplements of mineral nitrogen switch the lupine nutrition from the symbiotic to the autotrophic type, which is accompanied by a decrease in the nitrogen-fixing activity in the nodules and by an increase in the nitrate-reducing capacity of the roots and leaves.

Fabaceae↗

PubMed2006

Nitrogen fixation mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression.

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.

Cluster Analysis↗

PubMedDate not supplied

Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena.

Nitrogen fixation by the cyanobacterium Anabaena is carried out in heterocysts, specialized, non-dividing cells which differentiate under conditions of ammonia or nitrate deprivation. In Anabaena, heterocyst differentiation is accompanied by rearrangement of some nitrogen fixation genes. A site-specific recombination between an 11 base-pair direct repeat sequence flanking the nifK and nifD genes removes 11 kilobases of intervening DNA, resulting in juxtaposition of the two genes and an alteration of the nifD protein-coding sequence.

Cell Differentiation↗

PubMed1987

Regulation of soybean nitrogen fixation in response to rhizosphere oxygen: I. Role of nodule respiration.

Nitrogen fixation (acetylene reduction) rates of nodules on intact field-grown soybean (Glycine max) subjected to altered oxygen concentration (0.06-0.4 cubic millimeter per cubic millimeter) returned to initial rates during an 8-hour transitory period. Hydroponically grown soybean plants also displayed a transitory (1-4 hours) response to changes in the rhizosphere oxygen concentration after which the fixation rates returned to those observed under ambient oxygen concentrations. It was hypothesized that soybean nodules contain a regulatory mechanism which maintains a stable oxygen concentration inside nodules at a sufficiently low concentration to allow nitrogenase to function. A possible physiological mechanism which could account for this regulation is adjustment in nodule respiration activity such that nodule oxygen concentration and nitrogen fixation are maintained at stable levels. Experiments designed to characterize the non-steady-state oxygen response and to test for the presence of nodule respiratory control are presented. Non-steady-state acetylene reduction and nodule respiration (oxygen uptake) rates measured after alterations in the external oxygen concentration indicated that the regulatory mechanism required 1 to 4 hours to completely adjust to changes in the external oxygen concentration. Steady-state nodule respiration, however, did not respond to alterations in the rhizosphere oxygen concentration. It was concluded that soybean nodules can adjust to a wide range of rhizosphere oxygen concentrations, but the mechanism which controls nitrogen fixation rates does not involve changes in the nodule respiration rate.

Journal Article↗

PubMed1990

Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus.

Transcription of the genes that code for proteins involved in nitrogen fixation in free-living diazotrophs is typically repressed by high internal oxygen concentrations or exogenous fixed nitrogen. The DNA sequence of a regulatory locus required for repression of Rhodobacter capsulatus nitrogen fixation genes was determined. It was shown that this locus, defined by Tn5 insertions and by ethyl methanesulfonate-derived mutations, is homologous to the glnB gene of other organisms. The R. capsulatus glnB gene was upstream of glnA, the gene for glutamine synthetase, in a glnBA operon. beta-Galactosidase expression from an R. capsulatus glnBA-lacZ translational fusion was increased twofold in cells induced by nitrogen limitation relative to that in cells under nitrogen-sufficient conditions. R. capsulatus nifR1, a gene that was previously shown to be homologous to ntrC and that is required for transcription of nitrogen fixation genes, was responsible for approximately 50% of the transcriptional activation of this glnBA fusion in cells induced under nitrogen-limiting conditions. R. capsulatus GLNB, NIFR1, and NIFR2 (a protein homologous to NTRB) were proposed to transduce the nitrogen status in the cell into repression or activation of other R. capsulatus nif genes. Repression of nif genes in response to oxygen was still present in R. capsulatus glnB mutants and must have occurred at a different level of control in the regulatory circuit.

Base Sequence↗

PubMed1996

Problems and Promises of Assaying the Genetic Potential for Nitrogen Fixation in the Marine Environment

Nitrogen fixation in the sea has attracted the attention of ecologists for decades. Much is known about the habitats in which it occurs and some of the factors that limit N2 fixation activity in different environments, but we still know little about the organisms that fix nitrogen, and what limits the growth and distribution of these organisms in marine environments. Molecular biology technological developments have provided tools for detecting and characterizing N2-fixing organisms in the environment. These techniques hold great promise for unraveling the mysteries and paradoxes of N2 fixation in the sea. In this review, we address the theoretical basis for the use of a molecular approach to N2 fixation, highlight the strengths and weaknesses of the approach, and provide case studies that demonstrate the potential contribution of molecular biology approaches to studies of N2 fixation in the sea.

Journal Article↗

PubMed1994

Genetic regulation of nitrogen fixation in rhizobia.

This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans. Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54). The characteristic features of these proteins and their functions within species-specific regulatory pathways are described. Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA. FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL. In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription. This type of control includes FixLJ in R. meliloti and FixLJ-FixK in A. caulinodans or is brought about by autoregulation in B. japonicum. NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation. The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators. Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions. In B. japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.

Amino Acid Sequence↗

PubMed1976

Nitrogen fixation associated with grasses in Oregon.

Nitrogen fixation associated with both natural grasslands and grain crops of Oregon was studied using the acetylene-reduction assay. A number of the grasses collected has some acetylene-reducing activity. Agrostis tenuis Sibth. had substantially greater activity than any of the other species, with a mean rate estimated at 37 g N2 fixed per hectare per day. Assuming 100 days of activity, about 3 kg of N2 would be fixed per hectare per year. This quantity of nitrogen may be important in the maintenance of this species under natural conditions. Nitrogen-fixing microorganisms were isolated from the root surfaces of some of the grasses. Cultures of Bacillus macerans, Bacillus polymyxa, and Enterobacter cloacoa were isolated from wheat roots as were two cultures which have not been assigned a specific taxonomic classification. Strains of N2-fixing Bacillus species and Gram-negative aerobic bacteria were isolated from Festuca and Agrostis.

Acetylene↗

PubMed1997

Growth, nitrogen fixation, and nodularin production by two baltic sea cyanobacteria.

In late summer, nitrogen-fixing cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae form blooms in the open Baltic Sea. N. spumigena has caused several animal poisonings, but Baltic A. flos-aquae is not known to be toxic. In this laboratory study, performed with batch cultures, the influences of environmental conditions on the biomass and nitrogen fixation rate of N. spumigena and A. flos-aquae were compared and the toxin (nodularin) concentration produced by N. spumigena was measured. Several differences in the biomasses and nitrogen fixation rates of N. spumigena and A. flos-aquae were observed. A. flos-aquae preferred lower irradiances, salinities, and temperatures than N. spumigena. The biomass of both species increased with high phosphate concentrations and with accompanying bacteria and decreased with unnaturally high inorganic nitrogen concentrations. Nodularin concentrations in cells and growth media, as well as nitrogen fixation rates, were generally highest under the conditions that promoted growth. Intracellular nodularin concentrations increased with high temperature, high irradiance, and high phosphate concentration and decreased with low and high salinities and high inorganic nitrogen concentrations. Nodularin concentrations in growth media increased with incubation time, indicating that intracellular nodularin was released when cells lysed. The different responses of A. flos-aquae and N. spumigena to changes in salinity, irradiance, and temperature may explain the different spatial and temporal distribution of these species in the Baltic Sea. According to the results, toxic N. spumigena blooms may be expected in late summer in areas of the Baltic Sea with high phosphorus concentrations and moderate salinity.

Journal Article↗