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PubMed1980

Influence of various phenolic compounds on Azotobacter.

All the phenolic compounds, viz., nitrophenol, hydroquinone, coumarin, and benzilic acid, inhibited the growth, polysaccharide production, respiration, and nitrogen fixation of different Azotobacter isolates in vitro. Both in the presence and absence of various phenolic compounds a direct correlation between the polysaccharide production and nitrogen fixation was observed. However, no such direct effect was observed with respect to respiration and nitrogen fixation in the Azotobacter isolates tested.

Azotobacter↗

PubMed1998

Differential regulation of Rhizobium etli rpoN2 gene expression during symbiosis and free-living growth.

The Rhizobium etli rpoN1 gene, encoding the alternative sigma factor sigma54 (RpoN), was recently characterized and shown to be involved in the assimilation of several nitrogen and carbon sources during free-living aerobic growth (J. Michiels, T. Van Soom, I. D'hooghe, B. Dombrecht, T. Benhassine, P. de Wilde, and J. Vanderleyden, J. Bacteriol. 180:1729-1740, 1998). We identified a second rpoN gene copy in R. etli, rpoN2, encoding a 54.0-kDa protein which displays 59% amino acid identity with the R. etli RpoN1 protein. The rpoN2 gene is cotranscribed with a short open reading frame, orf180, which codes for a protein with a size of 20.1 kDa that is homologous to several prokaryotic and eukaryotic proteins of similar size. In contrast to the R. etli rpoN1 mutant strain, inactivation of the rpoN2 gene did not produce any phenotypic defects during free-living growth. However, symbiotic nitrogen fixation was reduced by approximately 90% in the rpoN2 mutant, whereas wild-type levels of nitrogen fixation were observed in the rpoN1 mutant strain. Nitrogen fixation was completely abolished in the rpoN1 rpoN2 double mutant. Expression of rpoN1 was negatively autoregulated during aerobic growth and was reduced during microaerobiosis and symbiosis. In contrast, rpoN2-gusA and orf180-gusA fusions were not expressed aerobically but were strongly induced at low oxygen tensions or in bacteroids. Expression of rpoN2 and orf180 was abolished in R. etli rpoN1 rpoN2 and nifA mutants under all conditions tested. Under free-living microaerobic conditions, transcription of rpoN2 and orf180 required the RpoN1 protein. In symbiosis, expression of rpoN2 and orf180 occurred independently of the rpoN1 gene, suggesting the existence of an alternative symbiosis-specific mechanism of transcription activation.

Amino Acid Sequence↗

PubMed2002

In vitro studies on the effects of herbicides on the growth of rhizobia.

AIMS: To study the possible adverse effect of herbicides on nodulation and nitrogen fixation in legumes by affecting the nitrogen-fixing rhizobia. METHODS AND RESULTS: Experiments were conducted to study the effect of four herbicides (terbutryn/terbuthylazine, trietazine/simazine, prometryn and bentazone) on the growth of nitrogen-fixing pea rhizobia (Rhizobium leguminosarum) in vitro by measuring optical density. Terbutryn/terbuthylazine, trietazine/simazine and prometryn had an adverse effect on the growth of rhizobia whereas bentazone was safe to rhizobia. CONCLUSIONS: The above herbicides could be used in pea at the recommended rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The adverse effects of herbicides on rhizobia were observed at concentrations not normally expected to occur under field conditions. Further, previously observed adverse effects of these herbicides on nodulation and nitrogen fixation of peas were, possibly, not due to their effects in rhizobia but to their adverse effects on the plant growth itself.

Culture Media↗

PubMed1997

Cloning, sequencing, and regulation of the global nitrogen regulator gene ntcA in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain BH68K.

In cyanobacteria, ammonium represses expression of proteins involved in nitrogen fixation and assimilation. The global nitrogen regulator gene ntcA encodes a DNA-binding protein, NtcA, that is a transcriptional activator of genes subject to nitrogen control. We report the cloning and sequencing of the ntcA gene from a nitrogen-fixing unicellular cyanobacterium, Cyanothece sp. strain BH68K. The gene comprises 678 nucleotides, and the deduced NtcA protein contains 226 amino acids with a predicted molecular weight of 25,026. In addition, ntcA mRNA levels were measured in cells grown under different nitrogen regimes. Under nitrogen-fixing conditions, ntcA transcripts were weakly expressed. Furthermore, ntcA expression was diminished or inversely proportional to nifHDK expression. Conversely, ntcA expression increased in nitrate-grown cells, and a concentration-dependent increase was seen in ammonium-grown cells up to 1 mM NH4Cl. These results indicate that ntcA is involved more in nitrogen assimilation than in nitrogen fixation and also imply that the rhythmic expression of ntcA and nifHDK transcription may be under the control of a circadian clock.

Aerobiosis↗

PubMedDate not supplied

[Ethylmethane sulfonate induction of auxotrophic mutants of Rhizobium meliloti and their characteristics].

Ethylmethanesulfonate was used as a mutagen to induce auxotrophic mutants in Rhizobium meliloti L5-30. Survival of the culture depended on the period of time during which it was treated with the mutagen. Thirteen auxotrophic mutants were isolated as a result of screening 1404 clones, and their requirements in growtn factors were determined. Ethylmethanesulfonate induced mainly those mutants which required sulfur-containing amino acids. The activity of nitrogen fixation of the auxotrophic mutants was assayed by measuring the reduction of acetylene and the yield of lucerne. Mutants requiring methionine or cystein, cysteine or histidine, and adenine with thiamine were not effective whereas those requiring arginine, tryptophan, and thiamine were capable of nitrogen fixation. Among two methionine auxotrophs, one displayed the activity of nitrogen fixation and the other did not.

Ethyl Methanesulfonate↗

PubMed2006

[Study of morphological and symbiotic traits during the ontogeny of supernodular and hypernodular pea mutants].

Morphological (plant height and vegetative biomass amount) and symbiotic (number of nodules and nitrogenase activity) traits of six symbiotic pea mutants and the original cultivar Rondo were studied at different vegetation periods. Of the mutants studied, one (K10a) was supemodular and the remaining five (K1a, K2a, K5a, K7a, and K27a) were hypemodular. Essential distinctions in the absolute values and time course of the changes in individual morphological and symbiotic traits of different pea mutants were demonstrated. The supemodular type is inferior to the original cultivar in plant height and production of vegetative biomass, but exceeds it in nodulation and nitrogen fixation. The hypemodular mutants either surpass the original cultivar with respect to the production capacity or display similar results. The symbiotic traits-number of nodules and nitrogen fixation activity--of these mutants are higher compared with the Rondo cultivar. The mutants K1a, K2a, and K27a were demonstrated to be useful in breeding pea for an increase in nitrogen fixation.

Biomass↗

PubMedDate not supplied

[Cloning and identification of the gene controlling nitrogen metabolism in the photosynthetic purple bacteria Rhodobacter sphaeroides].

A recombinant plasmid has been selected from the genomic library of Rhodobacter sphaeroides that restores the properties of the wild type strain in the mutant Drn121. The latter possesses the derepressed synthesis of nitrogenase when grown in the light, inability of nitrogen fixation in the dark and growth on potassium nitrate as a single source of nitrogen, disruption of ammonium ions and methylamine transportation, decreased activity of glutamine synthetase. The gene complementing the drn121 mutation is localized within the EcoRI-HindIII fragment of Rhodobacter sphaeroides chromosome 2.25 kb in size. Analysis of the fragment nucleotide sequence has revealed the fragments with a high level of homology to regulatory genes ntrB (the 3'-end) and ntrC of Rhodobacter capsulatus. The plasmid pRCN102, containing the nifR3-ntrB-ntrC operon of Rhodobacter capsulatus, is able to complement the drn121 mutation while its derivatives having inactivated ntrN or ntrC genes are not. Hence, in Rhodobacter sphaeroides mutant Drn121 the mutation is localized in ntrC gene the product of which is involved not only in nitrogen fixation but also in nitrogen metabolism on the whole.

Chromosome Mapping↗

PubMed1996

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae.

The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA. Results showed that a balanced synthesis of both gene products is essential for correct regulation. Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the phi10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA. In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low. This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL. Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA. Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes.

Bacterial Proteins↗

PubMed1999

Genetics of nitrogen regulation in Methanococcus maripaludis.

We have used genetic methods in Methanococcus maripaludis to study nitrogen metabolism and its regulation. We present evidence for a "nitrogen regulon" in Methanococcus and Methanobacterium species containing genes of nitrogen metabolism that are regulated coordinately at the transcriptional level via a common repressor binding site sequence, or operator. The implied mechanism for regulation resembles the general bacterial paradigm for repression, but contrasts with well-known mechanisms of nitrogen regulation in bacteria, which occur by activation. Genes in the nitrogen regulons include those for nitrogen fixation, glutamine synthetase, (methyl)ammonia transport, the regulatory protein GlnB, and ammonia-dependent NAD synthetase, as well as a gene of unknown function. We also studied the function of two novel GlnB homologues that are encoded within the nif gene cluster of diazotrophic methanogens. The phenotype resulting from a glnB null mutation in M. maripaludis provides direct evidence that glnB-like genes are involved in "ammonia switch-off," the post-transcriptional inhibition of nitrogen fixation upon addition of ammonia. Finally, we show that the gene nifX is not required for nitrogen fixation, in agreement with findings in several bacteria. These studies illustrate the utility of genetic methods in M. maripaludis and show the enhanced perspective that studies in the Archaea can bring to known biological systems.

Amino Acid Sequence↗

PubMed2005

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer.

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.

Aerobiosis↗

PubMed1994

Uncoupling of photosystems during light dependent dinitrogen fixation by a non-heterocystous cyanobacterium Plectonema boryanum.

Plectonema boryanum grows under microaerobic nitrogen starvation conditions by temporal separation of the oxygen-sensitive nitrogen fixation and the oxygen-evolving photosynthesis. During the nitrogen-fixation phase, depression in light-dependent oxygen evolution results from alterations in the stare of QA/QB complex. The reaction centre in photosystem II is not oxidised efficiently. Light dependence of nitrogenase function under such conditions appears to be related to the coupling of photosystem I to endogenous electron donors, such as glycollate. This cyanobacterium provides a natural example of in vivo shift in coupling between the photosystems during nitrogen-fixing growth.

Cyanobacteria↗

PubMed1992

Genetics of competition for nodulation of legumes.

An economically important problem in microbial ecology concerns the efficacy of rhizobial inoculants for the formation of nitrogen-fixing root nodules on legume crop plants such as soybean, alfalfa, and clover. Some strains of rhizobia can increase symbiotic nitrogen fixation under controlled conditions. However, attempts to improve nitrogen fixation under agricultural conditions with such strains often fail, usually as a result of the presence of indigenous rhizobia limiting nodulation by the inoculum strains. This problem is referred to as the Rhizobium competition problem, and molecular genetics is being used to address the problem from two perspectives. First, the host specificity of rhizobia is being characterized with the long term goal of developing strains that can nodulate a very strain-specific host-legume genotype. Second, the genetic basis of competitiveness in several strains is being examined. Genetic determinants of nodulation competitiveness have been isolated and mechanisms for their stable integration into the genome of superior nitrogen-fixing strains have been developed. Several phenotypes have been identified as playing an important role in nodulation competitiveness including antibiosis, motility, speed of nodulation, cell-surface characteristics, and nodulation efficiency. Several solutions to this problem are likely to result from these strategies and will be useful for certain legumes in specific locations.

Fabaceae↗

PubMed1999

Inhibition of the FixL sensor kinase by the FixT protein in Sinorhizobium meliloti.

Nitrogen fixation in symbiotic rhizobia is subject to multiple levels of gene regulation. In Sinorhizobium meliloti, the alfalfa symbiont, the FixLJ two-component regulatory system plays a major role in inducing nitrogen fixation and respiration gene expression in response to the low ambient O(2) concentration of the nodule. Here we report on the mode of action of the FixT protein, a recently identified repressor of nitrogen fixation gene expression in S. meliloti. First, we provide evidence that FixT prevents transcription of the intermediate key regulatory genes nifA and fixK by counteracting the activity of the FixLJ two-component system under otherwise inducing microoxic conditions. Second, we demonstrate that FixT acts as an inhibitor of the sensor hemoprotein kinase FixL, preventing the production or the accumulation of its phosphorylated form. FixT is thus a new example of a regulatory protein that blocks signal transduction in two-component systems at the level of the sensor kinase.

Bacterial Proteins↗

PubMed2006

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula.

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.

14-3-3 Proteins↗

PubMed2003

mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil.

The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH(4)NO(3) were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r(2) = 0.72) and in liquid culture (r(2) = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.

Azotobacter vinelandii↗

PubMed1996

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue.

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R, leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix+ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a flxK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified down-stream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.

Amino Acid Sequence↗

PubMed2004

Determination of nitrogen-fixing phylotypes in Lyngbya sp. and Microcoleus chthonoplastes cyanobacterial mats from Guerrero Negro, Baja California, Mexico.

In many environments, biological nitrogen fixation can alleviate nitrogen limitation. The high rates of N(2) fixation often observed in cyanobacterial mats suggest that N(2) fixation may be an important source of N. In this study, organisms expressing nifH were identified in a Lyngbya sp.- and two Microcoleus chthonoplastes-dominated cyanobacterial mats. The pattern of nitrogenase activity was determined for the Lyngbya sp. mat and a Microcoleus chthonoplastes mat sampled directly in Guerrero Negro, Mexico. Their maximum rates were 23 and 15 micro mol of C(2)H(4) m(-2) h(-1), respectively. The second Microcoleus mat, which was maintained in a greenhouse facility, had a maximum rate of 40 micro mol of C(2)H(4) m(-2) h(-1). The overall diel pattern of nitrogenase activity in the three mats was similar, with the highest rates of activity occurring during the dark period. Analysis of nifH transcripts by reverse transcription-PCR revealed that several different organisms were expressing nifH during the dark period. nifH phylotypes recovered from these mats were similar to sequences from the unicellular cyanobacterial genera Halothece, Myxosarcina, and Synechocystis, the filamentous cyanobacterial genera Plectonema and Phormidium, and several bacterial nifH groups. The results of this study indicate that several different organisms, some of which were not previously known to fix nitrogen, are likely to be responsible for the observed dark-period nitrogenase activity in these cyanobacterial mats.

Base Sequence↗

PubMed2001

Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti.

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the "ntrPR operon," which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

Amino Acid Sequence↗