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Results for “Symbiotic nitrogen fixation”

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At least 19 recordsLinked to original sources

PubMed1992

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation.

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates into bacteroids in the nodule for use as a carbon and energy source. In an attempt to increase dicarboxylate transport, a plasmid was constructed in which the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional dctA gene that was no longer under the control of the dctBD regulatory genes, but the recombinant plasmid was found to be unstable in R. meliloti. To stably integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that found in fully induced wild-type cells. Uptake assays showed that [14C]succinate transport by the trpPO-dctA fusion strain was constitutive, and the transport rate was the same as that of induced control cells. Acetylene reduction assays indicated a significantly higher rate of nitrogen fixation in plants inoculated with the trpPO-dctA fusion strain compared with the control. Despite this apparent increase, the plants had the same top dry weights as those inoculated with control cells.

Bacterial Proteins↗

PubMed1977

Energy requirement for symbiotic nitrogen fixation.

Comparing the energy required by legumes for symbiotic nitrogen fixation with that of assimilation of nitrate, Gibson concluded the costs to be about the same. About 15% of net photosynthetic production by the plant may be used in meeting its nitrogen requirements. If energy for the fixation of nitrogen symbiotically and that for the assimilation of NH4+ or NO3- from the soil solution are both provided by the chemical products of photosynthesis, then the CO2 respired in supplying that energy must contribute to the total efflux of CO2 from the plant in the dark. McCree and Thornley have discussed methods of partitioning the dark CO2 efflux into a growth (synthesis) and a maintenance component. Both nitrogen fixation and nitrogen assimilation can be expected to contribute to the CO2 flux associated with synthesis. If Gibson is correct, nodulated plants using only symbiotically fixed nitrogen should have the same growth coefficient as non-nodulated plants supplied with exogenous mineral nitrogen when grown in the same conditions. I have examined this hypothesis using a modification of the method used by McCree with Trifolium subterraneum L. cultivar Woogenellup as test material.

Carbon Dioxide↗

PubMed2005

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

Amino Acid Sequence↗

PubMed1982

Molecular cloning of Rhizobium trifolii genes involved in symbiotic nitrogen fixation.

DNA sequences responsible for the development and maintenance of symbiotic nitrogen fixation have been identified and isolated from Rhizobium trifolii. Symbiotically-defective strains were generated by random mutagenesis with the transposon Tn5. The defective genes which give rise to the mutant phenotype have been cloned into bacterial plasmids and used as hybridization probes to isolate the corresponding wild-type genes from a clone bank of R. trifolii DNA. Symbiotic genes cloned in this manner are able to correct the lesion caused by the insertion of the transposon in their respective mutants and so restore the nitrogen fixation phenotype. The correction of the mutation is shown to occur by two distinguishable mechanisms--either by complementation or by homologous recombination. This approach provides a reliable method for isolation and mapping of bacterial DNA sequences involved in symbiotic nitrogen fixation.

Cloning, Molecular↗

PubMed1977

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti.

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.

Ammonia↗

PubMed1996

Symbiotic nitrogen fixation does not require adenylylation of glutamine synthetase I in Rhizobium meliloti.

Symbiotic nitrogen fixation is accompanied by a shift of Rhizobium nitrogen metabolism from ammonium assimilation to ammonium export, which probably involves genetic or metabolic regulation of glutamine synthetase activity. In free-living Rhizobium meliloti glutamine synthetase I (GSI) is regulated post-translationally by reversible adenylylation in response to ammonium addition. Moreover, full expression of the GSI gene glnA requires the transcriptional activator, NtrC. A glnA1 mutant synthesizing a non-adenylylatable GSI produces normal nitrogen-fixing nodules on alfalfa: GSI adenylylation is dispensable for symbiotic nitrogen fixation. This is rationalized by the observation that less GS protein is present in R. meliloti bacteroids than in free-living bacterial cells.

Adenosine Phosphosulfate↗

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↗

PubMed1995

Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms.

Of the approximately 380 families of angiosperms, representatives of only 10 are known to form symbiotic associations with nitrogen-fixing bacteria in root nodules. The morphologically based classification schemes proposed by taxonomists suggest that many of these 10 families of plants are only distantly related, engendering the hypothesis that the capacity to fix nitrogen evolved independently several, if not many, times. This has in turn influenced attitudes toward the likelihood of transferring genes responsible for symbiotic nitrogen fixation to crop species lacking this ability. Phylogenetic analysis of DNA sequences for the chloroplast gene rbcL indicates, however, that representatives of all 10 families with nitrogen-fixing symbioses occur together, with several families lacking this association, in a single clade. This study therefore indicates that only one lineage of closely related taxa achieved the underlying genetic architecture necessary for symbiotic nitrogen fixation in root nodules.

Base Sequence↗

PubMed1989

Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation.

The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa). Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root. Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair. As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis. Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids. Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.

Journal Article↗

PubMedDate not supplied

Simulating interactive effects of symbiotic nitrogen fixation, carbon dioxide elevation, and climatic change on legume growth.

The underlying mechanisms of interaction between the symbiotic nitrogen-fixation process and main physiological processes, such as assimilation, nutrient allocation, and structural growth, as well as effects of nitrogen fixation on plant responses to global change, are important and still open to more investigation. Appropriate models have not been adequately developed. A dynamic ecophysiological model was developed in this study for a legume plant [Glycine max (L.) Merr.] growing in northern China. The model synthesized symbiotic nitrogen fixation and the main physiological processes under variable atmospheric CO2 concentration and climatic conditions, and emphasized the interactive effects of these processes on seasonal biomass dynamics of the plant. Experimental measurements of ecophysiological quantities obtained in a CO2 enrichment experiment on soybean plants, were used to parameterize and validate the model. The results indicated that the model simulated the experiments with reasonable accuracy. The R2 values between simulations and observations are 0.94, 0.95, and 0.86 for total biomass, green biomass, and nodule biomass, respectively. The simulations for various combinations of atmospheric CO2 concentration, precipitation, and temperature, with or without nitrogen fixation, showed that increasing atmospheric CO2 concentration, precipitation, and efficiency of nitrogen fixation all have positive effects on biomass accumulation. On the other hand, an increased temperature induced lower rates of biomass accumulation under semi-arid conditions. In general, factors with positive effects on plant growth tended to promote each other in the simulation range, except the relationship between CO2 concentration and climatic factors. Because of the enhanced water use efficiency with a higher CO2 concentration, more significant effects of CO2 concentration were associated with a worse (dryer and warmer in this study) climate.

Biomass↗

PubMed1992

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1982

Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes.

An 18 kb region adjacent to and surrounding the genes for nitrogenase (nif) was cloned from the genome of the symbiotic nitrogen-fixing species Rhizobium meliloti. A total of 31 Tn5 insertions in the nif region were constructed and assayed for their effect on symbiotic nitrogen fixation (Fix phenotype). Fix- insertions were found in two clusters, one 6.3 kb region not containing essential symbiotic genes. The locations of at least three transcription units containing Fix genes were deduced from complementation analysis between genomic nif::Tn5 insertions and nif::Tn5 insertions on mobilizable cloning vectors. The locations of R. meliloti genes nifH, nifD and nifK, which code for the single subunit of the nitrogenase Fe protein and for the two subunits of the nitrogenase MoFe protein respectively, were determined by DNA hybridization to cloned Klebsiella pneumoniae nif genes and by comparison of partial R. meliloti DNA sequences with K. pneumoniae nif gene sequences. R. meliloti nifH, D and K are located in the 6.3 kb fix-::Tn5 cluster and are transcribed in the order nifH, nifD, nifK, which is the same order as in K. pneumoniae.

Base Sequence↗

PubMed1983

Chromosomal gene controlling symbiotic nitrogen fixation in Rhizobium meliloti L5-30.

Auxotrophic Rhizobium meliloti strain RM 246 carries two independent mutations: in the biosynthesis of cysteine (cys) and symbiotic nitrogen fixation process (fix). These two mutations were mapped by transduction between his-240 and ade-4 markers. Cotransduction frequencies show the following order of genes: his-240 fix-1 cys-246 ade-4.

Chromosome Mapping↗

PubMed1998

Genes involved in the formation and assembly of rhizobial cytochromes and their role in symbiotic nitrogen fixation.

Rhizobia fix nitrogen in a symbiotic association with leguminous plants and this occurs in nodules. A low-oxygen environment is needed for nitrogen fixation, which paradoxically has a requirement for rapid respiration to produce ATP. These conflicting demands are met by control of oxygen flux and production of leghaemoglobin (an oxygen carrier) by the plant, coupled with the expression of a high-affinity oxidase by the nodule bacteria (bacteroids). Many of the bacterial genes encoding cytochrome synthesis and assembly have been identified in a variety of rhizobial strains. Nitrogen-fixing bacteroids use a cytochrome cbb3-type oxidase encoded by the fixNOQP operon; electron transfer to this high-affinity oxidase is via the cytochrome bc1 complex. During free-living growth, electron transport from the cytochrome bc1 complex to cytochrome aa3 occurs via a transmembrane cytochrome c (CycM). In some rhizobia (such as Bradyrhizobium japonicum) there is a second cytochrome oxidase that also requires electron transport via the cytochrome bc1 complex. In parallel with these cytochrome c oxidases there are quinol oxidases that are expressed during free-living growth. A cytochrome bb3 quinol oxidase is thought to be present in B. japonicum; in Rhizobium leguminosarum, Rhizobium etli and Azorhizobium caulinodans cytochrome d-type oxidases have been identified. Spectroscopic data suggest the presence of a cytochrome o-type oxidase in several rhizobia, although the absence of haem O in B. japonicum may indicate that the absorption attributed to cytochrome o could be due to a high-spin cytochrome b in a cytochrome bb3-type oxidase. In some rhizobia, mutation of genes involved in cytochrome c assembly does not strongly affect growth, presumably because the bacteria utilize the cytochrome c-independent quinol oxidases. In this review, we outline the work on various rhizobial mutants affected in different components of the electron transport pathways, and the effects of these mutations on symbiotic nitrogen fixation and free-living growth.

Copper↗

PubMed2006

Isocitrate dehydrogenase of Bradyrhizobium japonicum is not required for symbiotic nitrogen fixation with soybean.

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean (Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.

Arabinose↗

PubMed1993

Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for symbiotic nitrogen fixation is atypical.

In Rhizobium meliloti, an aspartate aminotransferase (AspAT) encoded within a 7.3-kb HindIII fragment was previously shown to be required for symbiotic nitrogen fixation and aspartate catabolism (V. K. Rastogi and R.J. Watson, J. Bacteriol. 173:2879-2887, 1991). A gene coding for an aromatic aminotransferase located within an 11-kb HindIII fragment was found to complement the AspAT deficiency when overexpressed. The genes encoding these two aminotransferases, designated aatA and tatA, respectively, have been localized by subcloning and transposon Tn5 mutagenesis. Sequencing of the tatA gene revealed that it encodes a protein homologous to an Escherichia coli aromatic aminotransferase and most of the known AspAT enzymes. However, sequencing of the aatA gene region revealed two overlapping open reading frames, neither of which encoded an enzyme with homology to the typical AspATs. Polymerase chain reaction was used to selectively generate one of the candidate sequences for subcloning. The cloned fragment complemented the original nitrogen fixation and aspartate catabolism defects and was shown to encode an AspAT with the expected properties. Sequence analysis showed that the aatA protein has homology to AspATs from two thermophilic bacteria and the eukaryotic tyrosine aminotransferases. These aminotransferases form a distinct class in which only 13 amino acids are conserved in comparison with the well-known AspAT family. DNA homologous to the aatA gene was found to be present in Agrobacterium tumefaciens and other rhizobia but not in Klebsiella pneumoniae or E. coli.

Amino Acid Sequence↗

PubMed2001

Regulation of gene expression in response to oxygen in Rhizobium etli: role of FnrN in fixNOQP expression and in symbiotic nitrogen fixation.

Previously, we reported finding duplicated fixNOQP operons in Rhizobium etli CFN42. One of these duplicated operons is located in the symbiotic plasmid (fixNOQPd), while the other is located in a cryptic plasmid (fixNOQPf). Although a novel FixL-FixKf regulatory cascade participates in microaerobic expression of both fixNOQP duplicated operons, we found that a mutation in fixL eliminates fixNOQPf expression but has only a moderate effect on expression of fixNOQPd. This suggests that there are differential regulatory controls. Interestingly, only the fixNOQPd operon was essential for symbiotic nitrogen fixation (L. Girard, S. Brom, A. Dávalos, O. Lopez, M. Soberón, and D. Romero, Mol. Plant-Microbe Interact. 13:1283-1292, 2000). Searching for potential candidates responsible for the differential expression, we characterized two fnrN homologs (encoding transcriptional activators of the cyclic AMP receptor protein [CRP]-Fnr family) in R. etli CFN42. One of these genes (fnrNd) is located on the symbiotic plasmid, while the other (fnrNchr) is located on the chromosome. Analysis of the expression of the fnrN genes using transcriptional fusions with lacZ showed that the two fnrN genes are differentially regulated, since only fnrNd is expressed in microaerobic cultures of the wild-type strain while fnrNchr is negatively controlled by FixL. Mutagenesis of the two fnrN genes showed that both genes participate, in conjunction with FixL-FixKf, in the microaerobic induction of the fixNOQPd operon. Participation of these genes is also seen during the symbiotic process, in which mutations in fnrNd and fnrNchr, either singly or in combination, lead to reductions in nitrogen fixation. Therefore, R. etli employs a regulatory circuit for induction of the fixNOQPd operon that involves at least three transcriptional regulators of the CRP-Fnr family. This regulatory circuit may be important for ensuring optimal production of the cbb(3), terminal oxidase during symbiosis.

Amino Acid Sequence↗

PubMed2003

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms.

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.

Carbon Dioxide↗