Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar `M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO₃ once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N₂. During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.     

Pathways of energy metabolism required for phenotypic expression of nif+Kp genes in Escherichia coli.

In E. coli K12 (F'nif+Kp) hybrids, electron-transport-dependent phosphorylation is not necessary for anaerobic nitrogen fixation, and substrate level phosphorylation can provide sufficient ATP from glucose for nitrogenase activity. The fumarate-reduction system, however, is essential in these hybrids for the transfer of electrons to nitrogenase. This system is probably also involved in maintaining the membrane in the energized state, thereby allowing nitrogen fixation to occur. The nitrate-reduction system, which can energize the membrane like the fumarate-reduction system, is not necessary for nitrogenase activity in the E. coli K12(F'nif+Kp) hybrids. However, two nitrate reductase genes, chlA, and chlB, are essential for inhibition of nitrogen fixation by nitrate. Moreover, nitrate inhibits nitrogenase activity and this inhibition is most probably effected through a regulator factor coded by chlA and chlB.     

Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation.

The soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.     

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.     

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.     

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 concentrations.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.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Physiological and agrochemical properties of different symbiotic genotypes of pea (Pisum sativum L.)

Physiological characters of symbiotic mutants of pea were studied: nodulation, activities of nitrogenase and nitrate reductase, chlorophyll content in leaves and their water-holding capacity, biomass accumulation, and nitrogen forms. The parameters reflecting the genotype state of the macrosymbiont under soil conditions considerably varied. Supernodulation mutants stood out against symbiotic pea genotypes by high contents of chlorophyll and nonprotein nitrogen compounds, high nitrogenase activity, and low nitrate reductase activity. The efficiency of the legume-rhizobium symbiosis was largely mediated by the macrosymbiont genotype. The highest atmospheric nitrogen fixation (50–80%) was observed in the parental pea varieties. Despite the highest nitrogenase activity in the nodules, the supernodulation mutants were inferior to the parental varieties by the nitrogen fixation capacity (40–60%), which was due to their low productivity.     

Nitrate Assimilation during Vegetative Regrowth of Alfalfa

Dry matter accumulation, nitrate reductase activity of various organs, nitrate accumulation, nitrogen derived from nitrate, and nitrogen content were studied during 17 days of vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.). Seedlings were grown in the glasshouse and treated with 0, 40, and 80 kilograms N per hectare applied as K¹⁵NO₃ to determine whether reduced nitrogenase activity after shoot harvest limited vegetative regrowth. The role of nodules in reducing NO₃⁻ during this period of low nitrogenase activity was also investigated.     

Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

Nitrate reductase and nitrogenase activities of common beans (Phaseolus vulgaris L.) from different geographic locations

Summary Cultivars ofPhaseolus vulgaris (L.) from contrasting geographic locations were cultivated under fields conditions for measurements of nitrogenase and nitrate reductase activities. A first trial with two cultivars indicated that a tropical cultivar B-789 has a higher nitrogenase activity than a temperate one Elsa. And the converse was true for the nitrate reductase activity. While where a post flowering application was made, a renewal of nitrate reductase activity occurred. Further similar comparisons of both enzymatic activities upon eight tropical and temperate cultivars of equivalent vegetative cycles indicated, on the average, that tropical cultivars have a higher level of (C₂H₂) reduction and a lower nitrate reductase activity than temperature cultivars. These observations suggest that there exists an inverse relationship between the two enzymatic activities in common beans, and there probably exists genetic variability for a possible improvement of N-fixation ability. An early application of N-fertiliser upon the Elsa and B-789 plots promotes later nitrogenase activity while a post flowering application shows obvious a renewal of nitrate reductase activity. Thus, analyses of nitrate reductase and nitrogenase activities of a common bean crop at different physiological stages may give us an indication of the best time to apply supplementary nitrogen fertilisation to common beans to increase seed yield.     

Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli.

Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.     

Possible causes of the physiological decline in soybean nitrogen fixation in the presence of nitrate

Nodulated soybean plants (Glycine max (L.) Merr. cv. Clarke)were supplied with 10 mol m^-3 nitrate at the vegetative stage.This treatment caused a rapid decline in nitrogen fixation (acetylenereduction) activity and a consequent decline in ureides in thexylem sap. However, there was virtually no effect on the nitrogenasecomplex, according to Western blots against components 1 and2. The effect on nitrogen fixation was matched by a decreasein nitrogenase-linked respiration and increases in nodule oxygendiffusion resistance and the carbon cost of nitrogen fixation.The addition of nitrate had little effect on protein contentfrom either nodule plant or bacteroid fractions. Activitiesof nitrate reductase (NR) and nitrite reductase (NiR) from eitherthe plant fraction or the bacteroids were affected in differentways during 8 d of supply. Nodule plant NR and bacteroid NiR were not affected. However,nodule plant NiR increased 5-fold within 2 d of supplying Bacteroid NR only increased after6 d. These results could be interpreted in terms of a restrictednitrate access into the infected region of nodules. However,denitrification was detected within 2 d of nitrate supply insoybean nodules. The results are discussed in relation to possiblecauses of the nitrate-induced decline in nitrogenase activity. Key words: Glycine max, nitrate, nitrogen fixation, nodules     

Variation in rates of nitrogen fixation in termites: response to dietary nitrogen in the field and laboratory

Abstract. Termites contribute nitrogen to their habitat through the nitrogenase activity of their bacterial symbionts. Previous studies indicate that high levels of dietary nitrogen suppress nitrogen fixation in termites. We examined the effects of dietary nitrogen on fixation rates in termites in both field and laboratory experiments. Ten field cplonies of Reticulitermes were collected and assayed for nitrogenase activity in July 1993, October 1993, January 1994, and April 1994. The nitrogen content of the wood collected with each colony was determined. There was no correlation between termite nitrogen fixation rates and the amount of nitrogen in their food for any of the four collection periods. In laboratory experiments, nitrogen fixation rates decreased when termites were fed filter paper treated with 2% and 5% ammonium nitrate or a 5% mixture of the amino acids proline, tryptophan and leucine, compared to water-treated controls. By contrast, the nitrogenase activity of termites fed filter paper treated with 2% and 5% ammonium phosphate, a mixture of the amino acids histidine, serine and aspartic acid, or 2% and 5% urea did not differ from the controls. However, nitrogenase activity increased when termites were fed with 2% uric acid. No clear association exists between termite nitrogen fixation and the nitrogen content of their food.     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Azide-resistant mutants ofAzorhizobium caulinodans with enhanced symbiotic effectiveness

Azide-resistant mutants ofAzorhizobium caulinodans strains Sb3, S78, SrR13 and SrS8 were isolated and screened for nitrate reductase activity. Selected nitrate reductase negative mutants were inoculated onSesbania bispinosa andS. rostrata under sterile conditions in chillum jars to study their symbiotic behavior. Azide-resistant mutants exhibited either similar or higher symbiotic effectiveness than the parent strain after 30 d of plant growth. Nodule mass, nitrogenase activity and uptake hydrogenase activity of the mutants varied depending on the host as well as on the plant growth stage. In comparison to wild-type parent strains, four azide-resistant mutants, Sb3Az18, S78Az21, SrR13Az17 and SrS8Az6 showed significant increase in nodulation and nitrogen fixation as well as shoot dry mass of the inoculated plants.     

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.     

Relationship between nitrogen fixation and nitrate metabolism in the Nodularia strains M1 and M2

Nitrogen fixation and nitrate-reduction activities were determined in photoautotrophic cultures of two wild-type strains of cyanobacterium Nodularia, spp. M1 and M2. Air could support growth of the two strains at a similar rate in the presence or absence of exogenous nitrate, ammonium and/or bicarbonate. Nitrogenase activity in air-grown cultures varied with culture age, and totally disappeared after 6 h of darkness. Recovery took place upon culture re-illumination. Ammonium at a concentration of 1 mM resulted in the total disappearance of nitrogenase activity and of heterocysts. In contrast, 20 mM nitrate hardly affected nitrogenase activity and heterocyst formation after ten generations. Under the same conditions, either ammonium or nitrate completely abolished nitrogenase activity and heterocyst formation in Anabaena sp. PCC 7119, a typical heterocystous strain. The inefficiency of nitrate in inhibiting nitrogen fixation in Nodularia M1 and M2 seemed to be caused by a low nitrate-reductase activity, and not by an impairment of nitrate-uptake activity. On the other hand, the presence of nitrate was not required for uptake activity to be expressed in Nodularia.Abbreviation NR nitrate reductase We thank C. Fernández-Cabrera (Consejo Superior de Investigaciones Científicas, CSIC, Madrid, Spain) for technical assistance, and Dr. G. Pérez-Silva (CSIC) for his collaboration in the Anabaena NR assays. This work was supported by grants from Spanish CI-CyT (PB 87-0204 and PB 92-0497).     

水稻根际兼性厌氧催娩克氏杆菌和阴沟肠杆菌的固氮与氢代谢

兼性厌氧细菌Enterobacter cloacae菌株E-26和Klebsiella oxytoca菌株NG-13的氢酶与固氮酶同时形成。固氮的最佳碳源为蔗糖、葡萄糖和丙酮酸,此外延胡索酸和苹果酸也能支持固氮。支持固氮的碳源也支持放氢,两者动力学基本一致。40％乙炔预处理后,吸氢活性下跌,放氢量未增加;NH_4~ 抑制固氮酶,但未导致放氢量降低;可能E-26菌株的放氢主要依赖于氢酶。菌株E-26和NG-13的吸氢反应,既能以O_2为电子受体,也能以延胡索酸、硝酸、MB为电子受体。但仅延胡索酸为电子受体时,E-26菌的固氮活性被分子H_2促进,它的氢吸收利用与固氮相偶联;而在CO_2和NH_4~ 代谢与H_2利用之间并无明显相关性,吸氢活性不被CO_2和NH_4~ 促进。