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1.
Growth and nitrogenase activity were studied in cultures of Azotobacter vinelandii growing with dinitrogen, ammonium sulfate, aspartic acid or yeast extract. Nitrogenase activity was measured by means of the C 2H 2 reduction test.In the presence of ammonium sulfate nitrogenase is completely repressed. After exhaustion of ammonia its activity is restored following a diauxic lag period of 30 min. With aspartic acid nitrogenase activity is partially repressed, and growth yield is higher than in the culture growing with N 2 only. This is due to simultaneous use of dinitrogen and aspartate. Fluctuations of nitrogenase activity occurring during exponential growth and the mechanism of their regulation are discussed.Abbreviations NA
nitrogenase activity
- BNF
Burk's nitrogen free medium 相似文献
2.
Biological nitrogen fixation (BNF), a key reaction of the nitrogen cycle, is catalyzed by the enzyme nitrogenase. The best studied isoform of this metalloenzyme requires molybdenum (Mo) at its active center to reduce atmospheric dinitrogen (N2) into bioavailable ammonium. The Mo-dependent nitrogenase is found in all diazotrophs and is the only nitrogenase reported in diazotrophs that form N2-fixing symbioses with higher plants. In addition to the canonical Mo nitrogenase, two alternative nitrogenases, which use either vanadium (V) or iron (Fe) instead of Mo are known to fix nitrogen. They have been identified in ecologically important groups including free-living bacteria in soils and freshwaters and as symbionts of certain cryptogamic covers. Despite the discovery of these alternative isoforms more than 40 years ago, BNF is still believed to primarily rely on Mo. Here, we review existing studies on alternative nitrogenases in terrestrial settings, spanning inland forests to coastal ecosystems. These studies show frequent Mo limitation of BNF, ubiquitous distribution of alternative nitrogenase genes and significant contributions of alternative nitrogenases to N2 fixation in ecosystems ranging from the tropics to the subarctic. The effect of temperature on nitrogenase isoform activity and regulation is also discussed. We present recently developed methods for measuring alternative nitrogenase activity in the field and discuss the associated analytical challenges. Finally, we discuss how the enzymatic diversity of nitrogenase forces a re-examination of existing knowledge gaps and our understanding of BNF in nature. 相似文献
3.
In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MB o) when the concentration of this mediator in the medium is increased up to 72 M. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NR o) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H 2 uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H 2 evolved/mg cell protein/min from the nitrogenase and 160 nM H 2-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H 2 evolution and for H 2 uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed. 相似文献
4.
Biological nitrogen fixation, the main source of new nitrogen to the Earth's ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo‐nitrogenase, the V‐nitrogenase and the Fe‐only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixation in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal‐replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo‐nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N 2 fixation in the same culture under metal limitation. We conclude that A. vinelandii is well adapted to fix nitrogen in metal‐limited soil environments. 相似文献
5.
Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N 2 reduction to NH 3 is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron‐molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo‐free nitrogenases that are less efficient at reducing N 2 than Mo‐nitrogenase. To permit biogenesis of Mo‐nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high‐affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo‐nitrogenase genes, while it represses production of Mo‐free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo‐mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo‐responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs. 相似文献
6.
15N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha -1 year -1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha -1 crop -1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. 15N 2 incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N 2-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha -1 crop -1. The problems associated with the use of the 15N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N 2-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N 2-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N 2-fixing systems in wetland rice and other cereal crops. 相似文献
7.
Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N 2 at 39° but grew normally at 30° on N 2 and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation. 相似文献
8.
Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co‐factors Mo and V. However, regulation of siderophore production by Mo‐ and V‐limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N 2‐fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe‐limitation the production of all siderophores increases, while under Mo‐limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo‐ and V‐limitation. An examination of the potential nitrogen ‘cost’ of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo‐ as opposed to the less efficient V‐nitrogenase. 相似文献
9.
Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H 2 evolution. The uptake hydrogenase was identified in all N 2-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N 2-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N 2-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H 2 production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl
chlorophyll
- MV
methyl viologen 相似文献
10.
Nitrogenase catalyzes not only the reduction of N 2 to NH 3 but also the reduction of C 2H 2 to C 2H 4 and H + ion to H 2 gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C 2H 2 reduction activity strongly but did not inhibit H 2 evolution activity. MA-2, on the contrary, inhibited only H 2 evolution activity. MA-8 inhibited both C 2H 2 reduction and H 2 evolution activity to the same extent. 相似文献
11.
Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. 相似文献
12.
It is generally reported that fungi like Pleurotus spp. can fix nitrogen (N 2). The way they do it is still not clear. The present study hypothesized that only associations of fungi and diazotrophs can
fix N 2. This was tested in vitro.
Pleurotus ostreatus was inoculated with a bradyrhizobial strain nodulating soybean and P. ostreatus with no inoculation was maintained as a control. At maximum mycelial colonization by the bradyrhizobial strain and biofilm
formation, the cultures were subjected to acetylene reduction assay (ARA). Another set of the cultures was evaluated for growth
and nitrogen accumulation. Nitrogenase activity was present in the biofilm, but not when the fungus or the bradyrhizobial
strain was alone. A significant reduction in mycelial dry weight and a significant increase in nitrogen concentration were
observed in the inoculated cultures compared to the controls. The mycelial weight reduction could be attributed to C transfer
from the fungus to the bradyrhizobial strain, because of high C cost of biological N 2 fixation. This needs further investigations using 14C isotopic tracers. It is clear from the present study that mushrooms alone cannot fix atmospheric N 2. But when they are in association with diazotrophs, nitrogenase activity is detected because of the diazotrophic N 2 fixation. It is not the fungus that fixes N 2 as reported earlier. Effective N 2 fixing systems, such as the present one, may be used to increase protein content of mushrooms. Our study has implications
for future identification of as yet unidentified N 2 systems occurring in the environment. 相似文献
13.
Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N 2 reduction by nitrogenase. To evaluate the significance of H 2 inhibition in vivo, we have measured the apparent inhibition constant for H 2 inhibition of N 2 reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N 2 reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O 2 and various concentrations of N 2 and H 2. The apparent inhibition constant for H 2 under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H 2 inhibition of N 2 reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules. 相似文献
14.
Gramineous crops such as wheat ( triticum oestivum), maize ( zea mays), and rice ( oryza sativa) develop tumorous structures (para-nodules) along primary and secondary roots when treated with low concentrations of various auxins. Rice forms additional tumors along its hypocotyle. Histologically, auxin-induced tumors appear as cancerous grown out root meristems and thus are comparable in origin and structure to stem nodules of the legume sesbania rostrata. Auxin-affected root meristems do not recover and develop further to large nodule-like organs. Introduced diazotrophs ( Azospirillum spp., Azorhizobium caulinodans, Rhizobium spp.) potentially inhabit tissues of both stem and root tumors with the central meristem as a major colonization niche. Evidence is given that infecting bacteria follow a ‘crack entry’ invasion at sites where developing tumors have emerged through the root cortex and epidermis. Bacteria are shown to establish with high cell numbers inside intercellular spaces of cortical and meristematic tissues. Plant-cell infection of tumor cells takes place with bacteria found inside the cell-cytoplasm surrounded by membrane-like structures. Once inhabiting induced tumor tissues introduced diazotrophs colonize endophytically with high cell numbers. Mutant, ammonium-excreting and thus ecologically disadvantaged A. brasilense is shown to survive inside para-nodulating maize and rice plants with a dense population. Micro-aerobic nitrogenase activities of tumor inhabiting diazotrophic bacteria ( A. brasilense, Azotobacter vinelandii, A. caulonidans) are in general highly increased when compared with untreated control plants. Additionally, bacterial nitrogenase activity is less sensitive to an increased oxygen tension in the root environment. The host plants benefit from the enhanced nitrogen fixation in their para-nodulating roots. Highest rates of incorporation of fixed nitrogen into host plant material is reported for para-nodule inhabiting ammonium excreting A. brasilense strain C3. The host plant potentially stimulates the nitrogenase activity of endophytically colonizing diazotrophs by providing energy in the form of a suitable carbon source. In conclusion, it is demonstrated that gramineous plants are potentially capable of developing an endophytical diazotrophic symbiosis through para-nodule formation. 相似文献
16.
Because of their ability to transform atmospheric N 2 into ammonia that can be used by the plant, researchers were originally very optimistic about the potential of associative diazotrophic bacteria to promote the growth of many cereals and grasses. However, multiple inoculation experiments during recent decades failed to show a substantial contribution of Biological Nitrogen Fixation (BNF) to plant growth in most cases. It is now clear that associative diazotrophs exert their positive effects on plant growth directly or indirectly through (a combination of) different mechanisms. Apart from fixing N 2, diazotrophs can affect plant growth directly by the synthesis of phytohormones and vitamins, inhibition of plant ethylene synthesis, improved nutrient uptake, enhanced stress resistance, solubilization of inorganic phosphate and mineralization of organic phosphate. Indirectly, diazotrophs are able to decrease or prevent the deleterious effects of pathogenic microorganisms, mostly through the synthesis of antibiotics and/or fungicidal compounds, through competition for nutrients (for instance, by siderophore production) or by the induction of systemic resistance to pathogens. In addition, they can affect the plant indirectly by interacting with other beneficial microorganisms, for example, Azospirillum increasing nodulation of legumes by rhizobia. The further elucidation of the different mechanisms involved will help to make associative diazotrophs a valuable partner in future agriculture. 相似文献
17.
Summary A cluster analysis based on codon usage in genes for biological nitrogen fixation ( nif genes) grouped diazotrophs into three distinct classes: anaerobes, cyanobacteria, and aerobes. In the nif genes of Klebsiella pneumoniae there was no evidence for selection pressure in favor of highly translatable codons. However, in the nitrogen regulatory operon glnAntrBntrC of enteric bacteria the stoichiometrically high level of glutamine synthetase may be facilitated by the presence of efficiently translatable codons in glnA. The nif genes of the cyanobacterium Anabaena showed codon selection in favor of translational efficiency. Computation of codon adaptation indices for expression in heterologous systems indicated that the reading frames most suitable for expression of nif genes in Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae were present in azotobacters, clostridia, and cyanobacteria, respectively. In codon-usage-based cluster analysis, type 3 nitrogenase genes of Azotobacter vinelandii grouped along with type 1 and type 2 genes. This is in contrast to the nucleotide sequence-based multiple alignment in which type 3 nitrogenase genes of A. vinelandii have been reported to cluster with entirely unrelated diazotrophs such as methanogens and clostridia. This may be indicative of lateral transfer of nif genes among widely divergent taxons. The chromosomal- and plasmid-located nif genes of rhizobia also cluster separately in nucleotide sequence-based analysis but showed similar codon usage. These analyses suggested that the phylogeny of nif genes drawn on the basis of nucleotide sequence homology was not masked by the taxon-specific pressure on codon usage. 相似文献
18.
Phototrophic diazotrophs include the photosynthetic green and purple bacteria, the heliobacteria, many cyanobacteria and the unusual chlorophyll-containing rhizobia that are found in the stem nodules of Aeschynomene spp. In this review, which concentrates on cyanobacteria, the interrelations between photosynthesis and N 2 fixation are discussed. Photosynthesis can, in theory, directly provide the ATP and reductant needed to support N 2 fixation but the link between these two processes is usually indirect, mediated through accumulated carbon reserves. In cyanobacteria, which possess an oxygenic photosynthesis, this serves to separate the O 2 that is produced by photosynthesis from the O 2-sensitive nitrogenase. However, in certain circumstances, oxygenic photosynthesis and N 2 fixation coexist. Under these conditions, respiratory consumption of photosynthetically generated O 2 may have an important role in minimizing O 2-damage to nitrogenase. 相似文献
19.
Ammonia (NH 3), an important raw material for chemical industry and agriculture, is also considered to be an intriguing energy storage and transportation media for chemical conversion schemes. The world's primary NH 3 supply is based on the natural nitrogen fixation by diazotrophs through an enzymatic nitrogenase process and the industrial nitrogen fixation through a traditional Haber–Bosch process. The natural synthesis of NH 3 can hardly meet the rapidly growing global demand. Meanwhile, the industrial NH 3 production is still dominated by the high‐temperature and high‐pressure reaction between nitrogen and hydrogen (N 2 + 3H 2 → 2NH 3), requiring intensive energy input and generating massive CO 2. Therefore, seeking a breakthrough in the development of catalysts toward efficient ammonia synthesis has become the frontier of energy and chemical conversion schemes. This review summarizes and discusses the recent progress on developing new strategies to optimize the efficiency of NH 3 production coupled with renewable energy sources, with a specific focus on electrocatalytic and photoelectrocatalytic conversion of N 2 to NH 3. The most recent advances in the development of catalytic materials, the design of the reaction systems, and the computational insights for electrochemical and photoelectrochemical ammonia synthesis are covered. 相似文献
20.
Biological nitrogen fixation (BNF) in woody plants is often investigated using foliar measurements of δ 15N and is of particular interest in ecosystems experiencing increases in BNF due to woody plant encroachment. We sampled δ 15N along the entire N uptake pathway including soil solution, xylem sap and foliage to (1) test assumptions inherent to the use of foliar δ 15N as a proxy for BNF; (2) determine whether seasonal divergences occur between δ 15N xylem sap and δ 15N soil inorganic N that could be used to infer variation in BNF; and (3) assess patterns of δ 15N with tree age as indicators of shifting BNF or N cycling. Measurements of woody N‐fixing Prosopis glandulosa and paired reference non‐fixing Zanthoxylum fagara at three seasonal time points showed that δ 15N soil inorganic N varied temporally and spatially between species. Fractionation between xylem and foliar δ 15N was consistently opposite in direction between species and varied on average by 2.4‰. Accounting for these sources of variation caused percent nitrogen derived from fixation values for Prosopis to vary by up to ~70%. Soil–xylem δ 15N separation varied temporally and increased with Prosopis age, suggesting seasonal variation in N cycling and BNF and potential long‐term increases in BNF not apparent through foliar sampling alone. 相似文献
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