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PubMed1977

Isolation and characterization of chlorate-resistant mutants of the blue-green alga Nosoc muscorum.

Spontaneous chlorate-resistant (Clr) mutants of three classes were isolated from Nostoc muscorum under three different selective conditions. A Clr-N2 class of mutants lacked nitrate reductase and showed nitrate inhibition of nitrogen fixation. A Clr-NO3 group of het+ nif- mutants formed heterocysts, but lacked nitrogen fixation and active nitrogenase enzyme. The Clr-NO2 class included those mutants deficient in both active nitrogenase and nitrate reductase, as they were unable to grow at the expense of molecular nitrogen or with nitrate nitrogen. The results suggest a common genetic determinant of active nitrogenase and nitrate reductase in the blue-green alga N. muscorum.

Chlorates↗
PubMed1987

Influence of the Bradyrhizobium japonicum Hydrogenase on the Growth of Glycine and Vigna Species.

The effect of the Bradyrhizobium japonicum hydrogenase on nitrogen fixation was evaluated by comparing the growth of Vigna and Glycine species inoculated with a Hup mutant and its Hup revertant. In all experiments, the growth of plants inoculated with the strain without hydrogenase was at least equal to the growth of the strain with hydrogenase. For Glycine usuriensis and Glycine max cv. Hodgson in liquid culture, the growth was higher with the Hup strain. It is possible that reduced rates of nitrogen fixation in the presence of hydrogenase are due to O(2) depletion caused by the hydrogen oxidizing, since the oxygen pressure in the air appears to be a limiting factor of symbiotic nitrogen fixation in the soybean.

Journal Article↗
PubMed2005

Effects of model root exudates on structure and activity of a soil diazotroph community.

Nitrogen fixation is often enhanced in the rhizosphere as compared with bulk soil, due to asymbiotic microorganisms utilizing root exudates as an energy source. We have studied the activity and composition of asymbiotic soil diazotrophs following pulse additions of artificial root exudates and single carbon sources, simulating the situation of bulk soil coming into contact with exudates from growing roots. Artificial root exudates and single sugars rapidly induced nitrogen fixation. The population of potential diazotrophs was studied using universal and group-specific nifH polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis. Reverse transcription PCR of nifH mRNA confirmed that phylotypes with an apparently increasing population size also expressed the nitrogenase system. According to our results, the actively nitrogen-fixing population represents only a fraction of the total diazotroph diversity, and the results of group-specific nifH PCR and phylogenetic analysis of cloned nifH and 16S rRNA gene fragments identified active species that belonged to the genus Azotobacter. Rapid changes of transcriptional activity over time were observed, indicating different growth and activation strategies in different Azotobacter strains. Only sugar-containing substrates were able to induce nitrogen fixation, but substrate concentration and the presence of organic acids may have additional selective effects on the active diazotroph population.

Acetic Acid↗
PubMed1983

Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti.

Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae. The host specificity of Rhizobium trifolii and R. leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid. The nodulation of white clovers was restored by either plasmid pJB5JI or pBR1AN when these plasmids were transferred to two transposon Tn5-induced hair-curling (Hac-) R. trifolii mutants. In addition, lucerne nodulation was restored to a Hac- R. meliloti mutant when either plasmid pBR1AN or pJB5JI was transferred to this strain. The phenotype of nonmucoid (Muc-) Rhizobium mutants, which had altered cell surfaces, was not influenced by the transfer to these strains of plasmid pBR1AN or plasmid pJB5JI.

Conjugation, Genetic↗
PubMed2025

ModE Regulates Alternative Nitrogenase Expression in the Methanogen Methanosarcina acetivorans.

All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans , also contain alternative vanadium- and iron-nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo-nitrogenase activity. The factors that control the expression of the alternative nitrogenases in response to Mo availability are unknown in methanogens. Here we show that ModE is the regulator that represses transcription of the vnf and anf operons in M. acetivorans when cells are grown with Mo. CRISPRi repression of modE results in a significant increase in the transcription of the vnf and anf operons as well as the detection of Fe-nitrogenase during nitrogen fixation in the presence of Mo. Gel shift assays with recombinant ModE demonstrated that ModE binds a specific sequence motif upstream of the vnf and anf operons, as well as other genes and operons related to nitrogen fixation and Mo transport. However, purified ModE does not contain Mo, and the addition of Mo does not alter the affinity of ModE for DNA, indicating M. acetivorans ModE may not directly bind Mo. This study shows that ModE is the primary Mo-responsive regulator of alternative nitrogenase expression in M. acetivorans , but other factor(s) are likely involved in directly sensing Mo.

Methanosarcina↗
PubMed1993

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.

Bacteria, Aerobic↗
PubMed1998

AIR synthetase in cowpea nodules: a single gene product targeted to two organelles?

A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.

Amino Acid Sequence↗
PubMed1997

Respiratory protection of nitrogenase activity in Azotobacter vinelandii--roles of the terminal oxidases.

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes--nitrogenases--are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.

Azotobacter vinelandii↗
PubMed1993

The C terminus of the alpha subunit of RNA polymerase is not essential for transcriptional activation of sigma 54 holoenzyme.

Several activators of sigma 70 holoenzyme whose binding sites lie upstream of the -35 region of promoters require the C-terminal region of the alpha subunit of RNA polymerase to activate transcription. (These are among class I activators, which require the C-terminal region of the alpha subunit for transcription activation.) Because transcription by sigma 54 holoenzyme universally depends upon activators whose binding sites lie well upstream (or downstream) of promoters, we determined whether the C-terminal region of the alpha subunit was also required for transcription from the sigma 54-dependent promoter for the glnA operon. Nitrogen regulatory protein C-dependent activation from the glnA promoter remained good when RNA polymerases containing C-terminal truncations of the alpha subunit were employed. This was also the case for nitrogen fixation protein A-dependent activation if a nitrogen fixation protein A-binding site was appropriately placed upstream of the glnA promoter. These results lead to the working hypothesis (as yet untested) that activators of sigma 54 holoenzyme, which appear to make direct physical contact with the polymerase to catalyze a change in its conformation, activate the sigma 54 holoenzyme by contacting the sigma subunit rather than the alpha subunit of the core enzyme.

Bacterial Proteins↗
PubMedDate not supplied

[Synthesis of the nitrogen-fixing enzyme system in oligonitrophilic bacteria].

Oligonitrophilic bacteria were cultivated on a medium containing only 2.5--10.0 mg/litre of nitrogen compounds. They assimilated elementary nitrogen only after utilization of these nitrogen compounds during growth and formation of nitrogen-fixing enzyme system. Their cells grown on a medium containing high concentrations of bound nitrogen did not fix nitrogen during further incubation in the atmosphere of 15N; therefore, the enzymes involved in nitrogen fixation were induced. These organisms are characterized by diauxia during their growth on media containing "start" doses of nitrogen. Enzymes catalysing nitrogen fixation in azotobacter are also induced. But, contrary to oligonitrophilic bacteria, the azotobacter does not require nitrogen compounds in the medium in order to adapt to molecular nitrogen, and its growth curve is not of a biphasal character. These data and the evidence of other authors suggest that all nitrogen-fixing microorganisms are characterized by the induced, not constitutive, enzymes involved in reduction of molecular nitrogen.

Bacillus↗
PubMed2000

Microdiversity of Culturable Diazotrophs from the Rhizoplanes of the Salt Marsh Grasses Spartina alterniflora and Juncus roemerianus.

Salt marshes dominated by Spartina alterniflora (smooth cordgrass) are among the most productive ecosystems known, despite nitrogen limitation. Rhizoplane/rhizosphere diazotrophy (nitrogen fixation) serves as a significant source of combined nitrogen in these systems. Several recent studies have demonstrated remarkable physiological and phylogenetic macro- and microdiversity within this important functional group of organisms. However, the ecological significance of this diversity is presently unknown. The physiological characteristics of the culturable, oxygen-utilizing fraction of the rhizoplane diazotroph assemblages from Spartina alterniflora and from another salt marsh grass, the black needle rush Juncus roemerianus, were examined in combination with an assessment of the phylogenetic relatedness by whole genome DNA-DNA hybridization. Analysis of substrate utilization data permitted quantitative evaluation of fully cross-hybridizing strain groups and physiological clusters. Phylogenetically related strains, defined by DNA homology >/=90% relative to the positive control, displayed extensive physiological diversity. Seven bootstrap-supported physiological clusters, composed largely of phylogenetically dissimilar strains, showed similar utilization patterns for at least one class of ecologically relevant substrates (carbohydrates, carboxylic acids, or amino acids). These diazotrophs appear to be physiologically adapted for utilization of specific substrates or classes of substrates, lending support to diazotrophic functional redundancy. Microenvironmental heterogeneity is credited for promoting this diversity by selecting for physiologically specialized diazotroph populations to occupy defined niches in situ. One outcome of this physiological diversity is maintenance of a crucial environmental function (nitrogen fixation) over a broad range of environmental conditions.

Journal Article↗
PubMed2004

The electron transfer flavoprotein fixABCX gene products from Azospirillum brasilense show a NifA-dependent promoter regulation.

The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric p fixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.

Azospirillum brasilense↗
PubMedDate not supplied

[The Rhizobium-Prosopis symbiosis in the Argentinian Chaco Arido].

Low productivity soils can be improved by incorporation of adapted legumes species able to increase the nitrogen fixation when associated to Rhizobium strains. Two fast-growing Rhizobium strains were isolated from nodules of Prosopis alba. Infectivity and effectivity of the strains were assessed under controlled conditions, measuring the following parameters: nodule number, dry weight, nitrogen percentage and fixed nitrogen. P. flexuosa strains showed high infective capacity (144 nodules/plant) but low effectivity (21.4 mg fixed nitrogen in 75 days). Although nodule number in P. alba was lower (23 nodules/plant) nitrogen fixation was higher (119.5 mg fixed nitrogen in 75 days) as compared with P. flexuosa. The isolated strains might be used as nitrogen donors in aride soils; they might be helpful to forestation of these ecosystems.

Argentina↗
PubMed2000

Three new NifA-regulated genes in the Bradyrhizobium japonicum symbiotic gene region discovered by competitive DNA-RNA hybridization.

The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613-622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoN mutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fix gene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded by nrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases and N-acetyltransferases, respectively. The product of nrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional -24/-12-type promoter upstream of nrgA and nrgBC and proved the NifA- and RpoN (sigma(54))-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery of nrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

Amino Acid Sequence↗
PubMed2005

Genotypic variation of N2-fixing common bean (Phaseolus vulgaris L.) in response to iron deficiency.

In calcareous soils, the yield of grain legumes is often limited by the lower availability of iron (Fe), especially when they depend upon symbiosis with root nodule bacteria for their N nutrition. In order to explore the variability of responses of N(2)-fixing common bean to Fe deficiency the common bean white-seeded lines Striker and Coco blanc, and coloured-seeded lines SVM-29-21 and ARA14 were inoculated with Rhizobium tropici (CIAT 899) and cultivated hydroaeroponically with a N-free nutrient solution supplied or not with 45microM Fe. Differences among lines were observed: Fe-deficiency-induced-chlorosis on young leaves was earlier and more severe in some lines than others. Nodule development and N(2)-fixing capacity was less affected in line ARA14 which preferentially allocated Fe towards nodules. Results suggest that Fe use efficiency for symbiotic nitrogen fixation (FeUE SNF) could be used to screen tolerant bean lines to Fe deficiency in condition of symbiotic nitrogen fixation.

Genetic Variation↗
PubMedDate not supplied

[Specific features of nitrogen transformation in the soddy-podzolic soil in the areas of habitat of the common vole Microtus arvalis].

The influence of common vole Microtus arvalis on processes of nitrogen fixation and denitrification in the soddy-podsolic soil was studied. In the common vole colonies, the level of nitrogen fixation was reliably lower and that of denitrification higher, than in the control soil outside these colonies. Nitrogen-containing excretory products of voles accumulating in the soil are among the main factors that determine the activity of these processes.

Animals↗
PubMed1995

Distinct and differently regulated Mo-dependent nitrogen-fixing systems evolved for heterocysts and vegetative cells of Anabaena variabilis ATCC 29413: characterization of the fdxH1/2 gene regions as part of the nif1/2 gene clusters.

Two different fdxH genes (fdxH1, fdxH2) have been isolated from the nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena variabilis ATCC 29413. They are part of two different nif gene clusters, nif1 and nif2. fdxH1 encodes the [2Fe-2S] ferredoxin that is known as the direct electron donor to nitrogenase in heterocysts, and is very similar to FdxH from Anabaena sp. PCC 7120. FdxH2 has more residues in common and shares its oxygen sensitivity with the single FdxH from the non-heterocystous, filamentous cyanobacterium Plectonema boryanum PCC 73110. The latter expresses nitrogenase early (< or = 3-4h) after nitrogen depletion in vegetative cells and exclusively under anaerobic conditions. fdxH2 and the nif2 genes of Anabaena 29413 are also transcribed < or = 4 h after onset of nitrogen-stepdown, exclusively under anaerobic growth conditions and long before functional heterocysts appear. At this time, no fdxH1 and nif1 gene transcription was observed. It occurred later and was associated with nitrogen fixation under aerobic conditions, i.e. within heterocysts. fdxH2 and nifHDK2 were not transcribed during aerobic, nitrogen-fixing growth. In addition, neither was an fdxH2-type gene found nor an anaerobically and early inducible Nif2 system detectable in Anabaena 7120. These data reveal that in filamentous cyanobacteria two different Nif systems have evolved based on molybdenum nitrogenases. It is concluded that a Nif2-type system operates in vegetative cells of non-heterocystous and some, but not all, heterocyst-forming filamentous cyanobacteria. It is environmentally regulated by the levels of both oxygen and combined nitrogen in the habitat. To simultaneously allow for oxygen-evolving photosynthesis and oxygen-sensitive nitrogen fixation, the Nif1-type system probably branched from an ancestral Nif2-type system and has evolved for an exclusive operation within heterocysts. Accordingly, its expression has become an obligate late event in the developmental programme of heterocyst differentiation, irrespective of aerobic or anaerobic growth conditions.

Amino Acid Sequence↗