Induction of nitrate assimilation in the cyanobacterium Anabaena variabilis

Filaments of Anabaena variabilis Kütz strain ATCC 29413 grown in the absence of nitrate contain nitrate reductase that is active in permeabilized filaments, but not in intact, living filaments until they have been incubated for about 40 min in the presence of nitrate. The delayed acquisition of the ability to reduce nitrate is insensitive to chloramphenicol. Thus, switching on of enzyme activity in the presence of nitrate does not involve protein synthesis and nitrate reductase activity is not regulated by the amount of enzyme present.     

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.     

Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70.

Klebsiella aerogenes W70 could grow aerobically with nitrate or nitrite as the sole nitrogen source. The assimilatory nitrate reductase and nitrite reductase responsible for this ability required the presence of either nitrate or nitrite as an inducer, and both enzymes were repressed by ammonia. The repression by ammonia, which required the NTR (nitrogen regulatory) system (A. Macaluso, E. A. Best, and R. A. Bender, J. Bacteriol. 172:7249-7255, 1990), did not act solely at the level of inducer exclusion, since strains in which the expression of assimilatory nitrate reductase and nitrite reductase was was independent of the inducer were also susceptible to repression by ammonia. Insertion mutations in two distinct genes, neither of which affected the NTR system, resulted in the loss of both assimilatory nitrate reductase and nitrite reductase. One of these mutants reverted to the wild type, but the other yielded pseudorevertants at high frequency that were independent of inducer but still responded to ammonia repression.     

Nitrate reductase and its role in nitrate assimilation in plants

Nitrate reductase (EC 1.6.6.1) is an enzyme found in most higher plants and appears to be a key regulator of nitrate assimilation as a result of enzyme induction by nitrate. The biochemistry of nitrate reductase has been elucidated to a great extent and the role that nitrate reductase plays in regulation of nitrate assimilation is becoming understood.     

Purification and characterization of acyl carrier protein from two cyanobacteria species

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.     

Nitrogen acquisition in four co-existing species from an upland acidic grassland

The concentration of both nitrate and ammonium nitrogen was measured in soil taken from an upland acidic (pH 4.5) grassland habitat, containing four co-existing species, Deschampsia flexuosa (L.) Trin., Festuca ovina L., Juncus squarrosus L. and Nardus stricta L. Both nitrate and ammonium nitrogen were found to be present in the soil, in similarly small quantities. The effect of both sources of nitrogen on relative growth rate was studied, and an attempt was made to determine whether nitrate or ammonium nitrogen is the immediate source of nitrogen for these plants using assays of nitrate reductase (EC 1.6.6.2) and ammonium uptake. All four species showed larger growth rates on the same concentration of ammonium nitrogen compared to nitrate nitrogen. All species showed low activities of leaf nitrate reductase, even in plants grown on 18 mol nitrate m⁻³. Ammonium uptake activity appeared to be higher in species which showed the lowest nitrate reductase activity and least response to increasing nitrate concentration in the growth medium.     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Effects of Energy Substrates on Nitrate Reduction and Nitrate Reductase Activity in a Ruminal Bacterium, Selenomonas ruminantium

The factors affecting the rate of nitrate reduction and the nitrate reductase content in Selenomonas ruminantium were examined. The rate of nitrate reduction per cell mass was higher when S. ruminantium was grown on lactate than when grown on glucose, and the rate was further enhanced when grown on succinate. The nitrate reduction rate was parallel to the nitrate reductase content in cells, suggesting that the amount of nitrate reductase limits the rate of nitrate reduction. The amount of nitrate reductase was inversely related to growth rate. The growth rate was related to the level of intracellular ATP, which was inversely related to the levels of ADP and AMP. The ratio of NADH to NAD⁺was related to the rate of nitrate reduction and to the amount of nitrate reductase. From these results, it is conceivable that the synthesis of nitrate reductase is regulated in response to the sufficiency of energy and electron supply. Intracellular concentrations of adenine nucleotides and pyridine nucleotides may be the regulating factors. The amount of nitrate reductase was increased by the presence of nitrate, suggesting that the synthesis of nitrate reductase is enhanced by nitrate. In addition, nitrate reduction altered the fermentation pattern as a result of electron consumption.     

Effect of nitrate and ammonium concentration on nitrate reductase activity in five species of mycorrhizal fungi

The nitrate reducing capacity of pure cultures of Cenococcum geophilum (Sow.) Ferd. & Winge, Paxillus involutes (Batsch: Fr.) Fr. (strains 1 and 2), Piloderma croceum Erikss. & Hjortst., Suillus variegatus (Fr.) O. Kuntze (strains 1 and 2) and an ectendomycorrhizal (E-strain) fungus was measured using an in vivo nitrate reductase (EC 1.6.6.3) assay. Differences between species and strains were established. The nitrate concentration of the culture medium influenced the nitrate reductase activities of the E-strain fungus and one strain of S. variegatus. The nitrate reductase activity of certain species and strains was a function of nitrate concentration. Addition of ammonium to the growth medium did not have any significant effect on the in vivo or in vitro nitrate reductase activity. The in vivo nitrate reductase activity in the mycelia of C. geophilum and the E-strain fungus decreased during 28 day growth in modified Melin-Norkrans medium. For mycelia of Paxillus involutus, Piloderma croceum and S. variegatus grown on agar the in vitro assays showed higher nitrate reductase activity than the in vivo assays.     

A comparison of inhibition of French-bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation

Inhibition by NO⁻₃ of acetylene reduction in bean ( Phaseolus vulgaris L. cv. Contender) and soybean ( Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C₂H₂ reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate-induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O²) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate-induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean.     

菠菜叶片中硝态氮还原与叶柄中硝态氮累积的关系

测定了不同生长期在不同施氮水平下3个菠菜品种各器官的硝态氮含量、叶片的硝酸还原酶活性、叶片细胞硝态氮的贮存库和代谢库大小.结果表明:叶柄中硝态氮含量远高于其它器官,其含量与叶片内源/外源硝酸还原酶活性的比值呈负相关;叶片细胞中硝态氮代谢库的大小与叶柄中硝态氮含量之间没有确定的关系.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.     

植物氮代谢硝酸还原酶水平调控机制的研究进展

氮代谢是植株体内最基本的物质代谢之一,硝酸还原酶是植物氮代谢的关键酶。主要对植物氮代谢在硝酸还原酶水平上调控的研究新进展,尤其是其合成／降解及活性调控机制进行了较为系统的综述。硝酸还原酶合成的调控主要发生在转录水平和翻译水平上,硝酸还原酶降解的调控主要发生在翻译后水平上,同时NO3^-及光在硝酸还原酶转录水平调控上的作用重大,硝酸还原酶编码基因转录的mRNA的稳定性强弱影响植物的氮代谢,而影响mRNA稳定性的因素很多,机理复杂;磷酸化／去磷酸化在硝酸还原酶活性调控中占举足轻重的地位,研究也比较深入。钝化蛋白也能够影响硝酸还原酶活性,许多小分子物质对硝酸还原酶活性有影响。     

Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719.

The effect of the nitrogen source on the cellular activity of ferredoxin-nitrate reductase in different cyanobacteria was examined. In the unicellular species Anacystis nidulans, nitrate reductase was repressed in the presence of ammonium but de novo enzyme synthesis took place in media containing either nitrate or not nitrogen source, indicating that nitrate was not required as an obligate inducer. Nitrate reductase in A. nidulans was freed from ammonium repression by L-methionine-D,L-sulfoximine, an irreversible inhibitor of glutamine synthetase. Ammonium-promoted repression appears therefore to be indirect; ammonium has to be metabolized through glutamine synthetase to be effective in the repression of nitrate reductase. Unlike the situation in A. nidulans, nitrate appeared to play an active role in nitrate reductase synthesis in the filamentous nitrogen-fixing strains Anabaena sp. strain 7119 and Nostoc sp. strain 6719, with ammonium acting as an antagonist with regard to nitrate.     

Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413

Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo-nitrogenase. The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth. Two of the three nitrogenases in this strain are Mo-dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain. High-affinity transport of molybdate in A. variabilis was mediated by an ABC-type transport system encoded by the products of modA and modBC. The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli. The deduced ModC part of the fused gene lacked a recognizable molybdate-binding domain. Expression of modA and modBC was induced by starvation for molybdate. Mutants in modA or modBC were unable to grow using nitrate or Mo-nitrogenase. Growth using the alternative V-nitrogenase was not impaired in the mutants. A high concentration of molybdate (10 microM) supported normal growth of the modBC mutant using the Nif1 Mo-nitrogenase, indicating that there was a low-affinity molybdate transport system in this strain. The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations. However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low-affinity molybdate transport system in this strain.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

A method for the determination of nitrate reductase

A procedure for the assay of nitrate reductase based on Szekely's diaminodiphenylsulphone method of nitrate determination (Szekely, E. (1967) Talanta 14, 941–950) is described. The method is simple and sensitive, allowing determination of 1 μg of nitrate in a volume of 1 ml or less. It is particularly suited to the determination of nitrate reductase.     

Alleviation of nitrate inhibition of soybean nodulation by high inoculum does not involve bacterial nitrate metabolism

Inoculation of soybean (Glycine max. cv. Bragg) plants with high level inoculum partially alleviated the nitrate inhibition of nodule formation (3 to 4 fold), but not nodule growth. This alleviation did not require the bacterial nitrate reductase asBradyrhizobium japonicum mutant strains 110CR1 and 110CR2 (both lacking assimilatory nitrate reductase activity) gave the same results as the wild type parent 311b110. The study was carried out in the glasshouse, thereby confirming preliminary field data by Herridgeet al. (1984) using a wild type bacterial inoculant.