Nitrogen metabolism

The proteolytic systems play an essential role in nitrogen metabolism of lactic acid bacteria in milk. The extracellular cell wall-bound proteinase is a key enzyme in this system, in which its activity is necessary for the growth of lactic acid bacteria in milk by initiating the breakdown of casein to smaller peptides. The screening of the proteolytic strains has been achieved on three solid media YMA, PCA and FSDA. The strain of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis, biovar. diacetylactis and the Enterococcus were tested. Lactococcus lactis produced a large hydrolysis zone more than 10 mm of diameter. The concentration of 3 and 5% of inoculum produced the highest acidity which was superior to 60°D. The proteolytic strains of Lactococcus lactis subsp. lactis biovar. diacetylactis at 3% of inoculum showed a maximal acidification rate 5°D /h and 0.3 unit pH/h. The strongest proteolytic strain Lactococcus lactis subsp. lactis biovar. diacetylactis produced ...

The development of drought tolerant genotypes for peanuts has now become a priority due to the growing number of drought-prone areas. The effects of drought stress on nitrogen metabolism was studied in leaves of two groundnut cultivars with differential sensitivity to drought stress,  K-134 (drought tolerant) and JL-24 (drought sensitive) subjected to different regimes of water stress conditions for a duration of 12 days. The total protein content in leaves of both cultivars declined with progressive accumulation of free amino acid levels. Concurrently, the protease activity in the tissues was also increased.  Ammonia content was increased in both cultivars and comparatively higher ammonia levels were recorded for cv. JL-24.  A gradual increase in the activities of key enzymes involved in nitrogen metabolism such as  glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate  dehydrogenase (NADH-GDH and NADPH-GDH), aspartate aminotransferase (AAT) and alanine aminotransferase (ALAT)  was observed in both cultivars subjected to water stress.  The increase in enzyme activities was more pronounced in the drought tolerant than in the drought sensitive cultivar. Contrarily, the activities of nitrate reductase (NR) and nitrite reductase (NiR) were decreased in the stressed plants. The extent of decrease was more in cv. K134 than cv. JL-24. The results indicate that drought tolerance of cultivar K-134 may be attributed at least in part to the ability to shift the metabolic rate leading to a greater accumulation of amino acids coupled with lower levels of ammonia and largely by reassimilation as evidenced by relatively greater activities of GS and GOGAT in the tissue. The physiological importance of enzyme alterations under water stress was investigated in relation to plant metabolism

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037- induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared...

The objective of this study was to evaluate the effect of filter cake biochar on the chemical and microbiological attributes of a dystrophic red-yellow latosol cultivated with macaúba (Acrocomia aculeata), araçá (Psidium firmum) and cajuzinho do cerrado (Anacardium humile), species native to the Cerrado. Thus, the responses of soil attributes were evaluated 120 days after transplanting the seedlings using four doses of biochar (1%, 2%, 4% and 8% v/v) and two control treatments (one with soil correction and fertilization and the other with no fertilization). The attributes evaluated were pH, contents of calcium, magnesium, phosphorus, potassium, CTC, total and microbial organic carbon and nitrogen, and C:N ratio. For the soils cultivated with the three Cerrado species, the 1% dose of biochar and mineral fertilization were the treatments that best conditioned the soil during the cultivation period and promoted a better response of the chemical and microbiological attributes of the soil.

The activity of glutamine synthetase from Aspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6•0-6•5, when the pH of the external medium was varied between 2•3-7•0. Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn 2+ ions partially protected the enzyme against this inactivation. Mn 2+dependent glutamine synthetase activity was higher at acidic pH (6•0) compared to Mg 2+supported activity. While the concentration of Mg 2+ required to optimally activate glutamine synthetase at pH 6•0 was very high (≥ 50 mM), Mn 2+ was effective at 4 mM. Higher concentrations of Mn 2+ were inhibitory. The inhibition of both Mn 2+ and Mg 2+ -dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the endproducts of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibited Aspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn 2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis.

The study of orchid mycorrhizal interactions is particularly complex because of the peculiar life cycle of these plants and their diverse trophic strategies. Here, transcriptomics has been applied to investigate gene expression in the mycorrhizal roots of Limodorum abortivum, a terrestrial mixotrophic orchid that associates with ectomycorrhizal fungi in the genus Russula. Our results provide new insights into the mechanisms underlying plant-fungus interactions in adult orchids in nature and in particular into the plant responses to the mycorrhizal symbiont(s) in the roots of mixotrophic orchids. Our results indicate that amino acids may represent the main nitrogen source in mycorrhizal roots of L. abortivum, as already suggested for orchid protocorms and other orchid species. The upregulation, in mycorrhizal L. abortivum roots, of some symbiotic molecular marker genes identified in mycorrhizal roots from other orchids as well as in arbuscular mycorrhiza, may mirror a common core of plant genes involved in endomycorrhizal symbioses. Further efforts will be required to understand whether the specificities of orchid mycorrhiza depend on fine-tuned regulation of these common components, or whether specific additional genes are involved.

The significance of nickel (Ni), which is essential for urease activity, for growth and nitrogen (N) metabolism of Brassica napus grown in nutrient solution with either NH % NO $ or urea as sole N source was investigated. Although Ni contents were below 25 µg kg -" d. wt, growth of plants relying on NH % NO $ was not affected by the Ni status. However, supplementing the growth medium with 0n04 µ Ni enhanced dry matter production of urea-grown plants significantly. Urease activity was significantly reduced in leaves and roots of plants grown without supplementary Ni irrespective of N source. Plants grown with urea without additional Ni accumulated large amounts of urea and had lower amino acid contents indicating impaired usage of the N supplied, while those grown with NH % NO $ under Nideprived conditions accumulated endogenous urea in their older leaves. It is suggested that Ni may not be strictly essential for plants receiving mineral N, or that the critical level is well below 25 µg kg -" d. wt. These results confirm that Ni is required for urease activity and thus for growth of plants on urea-based media, as well as for recycling endogenous urea.

Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-tocopherol and free amino acids (faa). The yield of metabolites is dependent on the strain of E. gracilis and the metabolic growth condition. Here we investigated the effects of photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on the synthesis of ascorbate, α-tocopherol and faa in E. gracilis var. saccharophila, using label-free shotgun proteomics, and metabolite analysis using colourimetric assay, high-performance and ultra-performance liquid chromatography (HPLC/UPLC). PT cultivation resulted in the production of more antioxidants (up to 4.13 mg g -1 ascorbate and 2.52 mg g -1 α-tocopherol) than the MT and HT growth conditions (up to 0.97 and 0.50 mg g -1 ascorbate, and 1.40 and 0.21 mg g -1 α-tocopherol, respectively). The relative abundance of several faa varied between mid-log and initial stationary growth phases, but the total amount of faa remained about the same, with arginine as the most abundant amino acid. Proteomic analysis revealed a total of 3843 nonredundant proteins in E. gracilis var. saccharophila, of which 1890 were common among all cultivations. Gene ontology annotations suggested derivatisation of metabolic pathways from different organisms, such as lysine biosynthesis from fungi and serine biosynthesis from plants, while a few pathways were unique to Euglena, such as those of ascorbate and arginine. Some enzymes exhibited several isoforms that were influenced by the metabolic growth condition. For example, one of the isozymes of threonine aldolase was expressed in HT/MT cultures only, and one of the isozymes of phosphoglycerate dehydrogenase was expressed in PT cultures only. This is the first proteomic study of E. gracilis var. saccharophila, which provides a mechanistic insight into the biosynthetic pathway dynamics of primary metabolites (antioxidants and faa). This new information can serve as a framework for further development of Euglena as a producer of nutraceuticals.

Ammonium supplied to Acer cells incubated in Tricine buffer raised net dark incorporation of C-bicarbonate by 1.5 to 3,3 fold; this stimulation was not abolished by prior inhibition of glutamine synthetase by methionine sulphoximine. With cells in phosphate buffer, ammonium gave a smaller 1,15 to 1,3 fold stimulation which was abolished when glutamine synthetase was inhibited. Ammonium had no direct effect on the activities of phosphoenolpyruvate carboxyiase and pyruvate kinase assayed in partially purified extracts or on the enzymes catalysing release of label.

Beggiatoa atba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 raM. B. aIba coupled in vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction. B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction by B. alba was ammonia; N2 or N20 were not produced. The nitrate reductase activity in B. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. The B. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of the B. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus, B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.

Sugar beet fertilization is more specific than other field crops, which is reflected in the fact that to achieve high sugar yield per unit area, with a high root yield is very important and its technological quality. Researches of impact of fertilization on these sugar beet traits in the period of three years are conducted in the stationary field experiment at the Institute of Field and Vegetable Crops in Novi Sad, Serbia, at Rimski Sancevi experimental station. The experiment was carried out on ten fertilization variants with increasing doses of N, P2O5 and K2O, and the object of investigation was a local variety Sara. Root yield was the highest on the fertilization variants with the highest amounts of nitrogen fertilizer, but in any case, the difference in yield was not statistically significant in comparison with variants in which it was applied 100 kg N ha -1 . The sugar content significantly decreased, while the content of harmful nitrogen and sodium significantly increased wit...

A commonly occurring auxin indole-3-aceticacid (IAA) and a rarely occurring chlorosubstituted auxin (4-Cl-IAA) were compared for their impact on growth and nitrogen metabolism in mung bean for the first time. The plants were generated from healthy and Rhizobium coated seeds in earthen pots. The seedlings at 7 and/or 14 days were percolated with 0, 10 210 , 10 28 , or 10 26 M of IAA or 4-Cl-IAA. The plants were sampled at 30 days after sowing (DAS) to assess the growth and various biochemical characteristics. The auxins significantly enhanced the growth (length and dry mass of shoot and root), nodule fresh mass, nitrogenase activity in fresh nodules, leaf carbonic anhydrase activity, chlorophyll content, and rate of photosynthesis. The effect of the auxins lasted up to the harvest where the seed yield, 100 seed mass, and number of pods per plant were significantly affected by the auxins. At a moderate concentration (10 28 M), 4-Cl-IAA generated the best response. However, a comparable response was generated by the higher concentration (10 26 M) of 4-Cl-IAA. The application of the hormone twice (at 7 and 14 DAS) was much more effective than single application (at 7 or 14 DAS). It was concluded that IAA and 4-Cl-IAA improved the growth and nitrogen fixation in mung bean. The 4-Cl-IAA proved more effective than IAA.

Transgenic tomato (Solanum lycopersicum) plants were generated expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in photosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency (Fv/Fm). Furthermore, a clear reduction in flux through the tricarboxylic acid (TCA) cycle was observed in the transformants. Additionally, biochemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD(P)H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochondrial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency.

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADPdependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F v /F m ), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.

Effect of NH, + and NO3-on XSN-alloeation to organs and nitrogenous compounds in potato plants (Solarium tuberosum L.) was examined in hydroponic culture using an automatic pH control system, and the results obtained were as follows. 1) Growth of upper leaves was stimulated by NH4 + because lSNH4-N was preferentially distributed into upper leaves compared to 15NOa-N which was mainly distributed to lower leaves. 2) At the vegetative growth stage, tSNH4-N and ~sNO3-N distribution among organs was almost similar except for roots, whereas at the tuber swelling stage, lSNH,-N was distributed to stems and roots, and ~sNOa-N was distributed to leaves. 3) 15N amino acid metabolism was affected by NH4 + or NOa-, for example, in leaves grown in ~SNH4-N, the contents of aspartie acid and asparagine (Asx) increased with growth, while those of glutamie acid and glutamine (Glx) decreased with growth. In leaves grown in tsNOa-N, the Asx content remained constant, but the Glx content increased with growth. In tubers, ISNH4-N was distributed to Asx at first, but ~NO~-N was distributed to Glx. Thus, the metabolism and translocation pattern of Asx and Glx were quite different depending on the nitrogen forms.

Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also in¯uences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid pro®les, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids.

A computer model of C 3 photosynthesis comprising light reactions, electron-proton transport, enzymatic reactions and regulatory functions is presented as a system of differential budget equations for intermediate compounds. Carbon and nitrite reduction systems are linked assuming that nitrite reduction is the

Transgenic tomato (Solanum lycopersicum) plants were generated expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in photosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency (Fv/Fm). Furthermore, a clear reduction in flux through the tricarboxylic acid (TCA) cycle was observed in the transformants. Additionally, biochemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD(P)H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochondrial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency.

summaryThe effect of infrequent drinking on nitrogen metabolism was studied in Bedouin goats, a breed herded in the extreme deserts of the Middle East. The goats were given water either daily or only once every 4 days, while being maintained on roughages of different quality: lucerne hay (19% protein), Rhodes grass (10% protein) or wheat straw (3·7%). On lucerne hay and Rhodes grass the goats kept their body mass constant, whether given water daily or only once every 4 days. When on wheat straw the body mass was maintained constant only when given water once every 4 days. Infrequent drinking was found to hamper considerably the food intake of the goats when fed on high quality roughage. When on the wheat straw, the nitrogen intake of the goats (only 12% of that recorded when fed lucerne hay and given water daily) was, however, not affected by the drinking regime. Infrequent drinking improved nitrogen digestibility on all the diets, but more so on those of low quality than on the luc...

Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37°°°°C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309± ± ± ±35 nkat mg -1 ) and GS1b (1,166± ± ± ±65 nkat mg -1 ) enzymes were obtained by decreasing the expression temperature to 10°°°°C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.

The activity of glutamine synthetase from Aspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6•0-6•5, when the pH of the external medium was varied between 2•3-7•0. Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn 2+ ions partially protected the enzyme against this inactivation. Mn 2+dependent glutamine synthetase activity was higher at acidic pH (6•0) compared to Mg 2+supported activity. While the concentration of Mg 2+ required to optimally activate glutamine synthetase at pH 6•0 was very high (≥ 50 mM), Mn 2+ was effective at 4 mM. Higher concentrations of Mn 2+ were inhibitory. The inhibition of both Mn 2+ and Mg 2+ -dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the endproducts of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibited Aspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn 2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis.

SUMMARYWe characterized the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in tissues of the gulf toadfish Opsanus beta subjected to unconfined(ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that mass-specific GSase activities rank in the order of brain> liver > stomach ≈ kidney > intestine > gill > heart/spleen> muscle. When tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine, with minor contributions from the remaining tissues. Additionally,during confinement stress, GSase activity increases significantly only in liver (fivefold) and muscle (twofold), tissues that previously showed significant expression of the other enzymes of urea synthesis. Western analyses of samples on SDS gels demonstrated that GSase-specific protein content reflected enzyme activity, ...

Field studies were conducted in Johnson Key Basin, Florida Bay, USA from September 2002 through September 2004 to examine physiological, ecological, and behavioral characteristics of the gulf toadfish, Opsanus beta (Goode and Bean in Proc US Natl Mes 3:333-345, 1880), in relation to nitrogen metabolism, habitat usage, and spawning. Fish collected 5 cm above sediments in experimental shelters (epibenthic) were compared with those collected by throw traps which were found on or burrowing within sediments. The relationship between microhabitat ammonia and urea excretion, as determined by the enzymatic activity of glutamine synthetase (GS), was examined. The hypothesis tested was that O. beta occupying epibenthic nests were less ureotelic with lower GS activities than non-nesting individuals on/in sediments, due to a decreased environmental ammonia burden. Porewater total ammonia (T Amm ) concentrations at a sediment depth of 5 cm, i.e., the approximate depth of burrowing toadfish, ranged from 0 to 106.5 lmol N l -1 while the pH ranged from 7.48 to 9.14. There was a weak but significant correlation between environmental ammonia (NH 3 ) concentration and hepatic GS activity for epibenthic toadfish (P < 0.001, r 2 = 0.10), but not for burrowing toadfish. Mean urea-N and T Amm concentrations within shelters occupied by toadfish (n = 281) were 9.8 ± 0.83 lmol N l -1 and 13.0 ± 0.7 lmol N l -1 , respectively. As predicted, hepatic GS activity was significantly lower in epibenthic toadfish captured in shelters (4.40 ± 0.24 lmol min -1 g -1 ; n = 281) as compared to individuals on/in sediments (6.61 ± 0.47 lmol min -1 g -1 ; n = 128). Glutamine synthetase activity generally peaked in March (spawning season) and was lowest in July. Gender differences in hepatic and branchial GS activity were also found during the spawning season, which is attributable to the fact that males brood and guard offspring in their epibenthic nests while females often rest on or burrow into the sediments. Finally, hepatic and branchial GS appeared to have different patterns of enzymatic activity suggesting functional differences in gene expression.

SUMMARYWe characterized the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in tissues of the gulf toadfish Opsanus beta subjected to unconfined(ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that mass-specific GSase activities rank in the order of brain&gt; liver &gt; stomach ≈ kidney &gt; intestine &gt; gill &gt; heart/spleen&gt; muscle. When tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle &gt; stomach and intestine, with minor contributions from the remaining tissues. Additionally,during confinement stress, GSase activity increases significantly only in liver (fivefold) and muscle (twofold), tissues that previously showed significant expression of the other enzymes of urea synthesis. Western analyses of samples on SDS gels demonstrated that GSase-specific protein content reflected enzyme activity, ...

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoPbinding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.

The aim of this study was to evaluate the biochemical events in root nodules which lead to increased yield when bean is inoculated with a Rhizobium etli mutant (CFN037) having increased respiratory capacity. CFN037-inoculated plants had 22% more nitrogen (N) than did wild-type (CE3)-inoculated plants. Root nodule enzymes involved in nodule carbon and nitrogen assimilation as well as in ureides and amides synthesis were assessed in plants inoculated with CFN037 and the CE3. Our results show that the xylem ureides content was lower while that of amino acids was higher in CFN037- compared with CE3-inoculated plants. Supporting these results, enzymes involved in ureide synthesis were reduced while activity of aspartate aminotransferase, glutamate synthase, sucrose synthase, and glucose-6-P dehydrogenase were increased in CFN037- induced nodules. Glutamate synthase and phosphoenolpyruvate carboxylase transcripts were detected early in the development of nodules induced by CFN037 compared...

This study evaluated the effects of dietary organic acid (OA) blend on hemato-immunological responses, reproduction capacity, gene expression, and histological features of red tilapia (Oreochromis niloticus × O. mossambicus) broodstock. Four diets were formulated, containing 0 (control), 2, 3, and 4 g OAs kg-1. The diets were fed to triplicate groups (n = 3) of red tilapia broodstock (75 ± 5.56 g((mean ± standard deviation (SD)) stocked in 10-m 3 concrete tanks at a male to female ratio of 1:3, to satiation, twice a day, for 8 weeks. At the end of the feeding trial, fish in each tank were collected, counted, and weighed. Blood samples were collected from five fish from each tank and used for

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of puriWed recombinant protein showed that the fungal gdhA had 5.4-10.2 times higher V max value and 15.9-43.1 times higher K m value for NH 4 + , compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimila-tion pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with 15 NH 4 + showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH 4 + absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when suYcient nitrogen was present, but did not do so under low-nitrogen conditions. Under Weld condition, the transgenic line examined showed a signiWcant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium. Rice • Aspergillus niger • NADP(H)dependent glutamate dehydrogenase • Nitrogen metabolism Abbreviations CE-MS Capillary electrophoresis-mass spectrometry GDH Glutamate dehydrogenase GOGAT Glutamate synthase GS Glutamine synthetase MSX Methionine sulfoximine 2-OG 2-Oxoglutarate

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15NH4Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with ...

The influence of different N sources on fermentation rate and de novo amino acid synthesis by rumen micro-organisms was investigated in vitro using rumen fluid taken from four sheep receiving a mixed diet comprising (g/kg DM): grass hay 500, barley 299.5, molasses 100, fish meal 91, minerals and vitamins 9.5. Pancreatic casein hydrolysate (P; comprising mainly peptides with some free amino acids; 10 g/l), free amino acids (AA; casein acid hydrolysate + added cysteine and tryptophan; 10 g/l), or a mixture of L-proline, glycine, L-valine and L-threonine (M; 0.83 g/l each) were added to diluted (1:3, v/v), strained rumen fluid along with 15NH4Cl (A; 1.33 g/l) and 6.7 g/l of a mixture of starch, cellobiose and xylose (1:1:1, by weight). P and AA, but not M, stimulated net gas production after 4 and 8 h incubation (P &lt; 0.05) in comparison with A alone. P increased microbial-protein synthesis (P &lt; 0.05) compared with the other treatments. All of the microbial-N formed after 10 h was...

Se describe la variación espacial de la materia orgánica particulada (MOP), en términos de proteínas, carbohidratos y clorofilas, así como la abundancia específica del titoplancton, para bahía Concepción durante el pico reproductivo de Argopecten circuluris. La distribucibn de estas variables evidenció dos zonas de mayor concentración de partículas: una en el primer "tercio" de la bahía y la otra en la cabecera. Los valores altos de clorofila a se debieron al nanofitoplancton, el cual contribuyó en mayor proporción a la abundancia total. Las especies abundantes del microtitoplancton fueron Chaetoceros curvisetus, Ch. lorenzianus, Ch. teres, Ch. cinctus y Lithodesmiun undulatum. El cálculo de las contribuciones de materia orgánica particulada fitoplanctónica y no fitoplanctónica mostró que en superficie el mayor porcentaje de proteínas (69.5%) está dado por el fitoplancton, mientras que a 10 m correspondió al material organito particulado no fitoplanctónico o detrítico. Esto permite inferir una alta calidad de la MOP en la columna de agua, la cual constituye una fuente potencial de alimento para los diferentes estadios de la almeja catarina.

The habituated callus is a vitrified tissue which has two main biochemical characteristics both leading to production of toxic forms of oxygen: first the blockage of the porphyrin pathway and a lack of H20 2 detoxifying enzymes (catalase and peroxidases); secondly a deviation of the nitrogen metabolism induced by NH 3 accumulation. Ammonia detoxification is ensured by increased glutamate dehydrogenase activity and accumulation of both proline and polyamines. A putative linkage between proline synthesis and the HMP pathway, as proposed for animal proliferating tissues , might explain a high purine biosynthesis and cytokinin autonomy.

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll-deficient callus, was linked to accumulation of proline and polyamines. Experiments with a-difluoromethylornithine (DEMO) and a-difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H calius was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperbydric tissue.

Diatoms are the finest alternative to the natural feed stock of fuel and energy rich molecules. They are abundant in ecosystem and contains lipid as their primary reserved food material hence turns out to be a promising source of industrial fatty acids and bioactive compounds. Present study investigates potential applications of native diatom Nitzschia sp. isolated from Karnatak University Campus, Dharwad, further employed for the synthesis of lipids and high value fatty acids. This newly isolated diatom Nitzschia sp. cultivated by photoautotrophic cultivation method using f/2 growth media supplemented with NaNO3, trace metals and vitamin solution. Nitrogen concentration in the media gradually lowered to enhance lipid synthesis though batch culture. Nitrogen deficient culture showed increase in lipid productivity by Nitzschia sp. and total lipid yield obtained is 05.751mg/L. The GC-MS analysis indicates n-hexane lipid fraction of test diatom mainly composed of decosahexaenoic acid C...

Twenty four primiparous and multiparous Holstein cows on early lactation, beginning five weeks postpartum, were used for four weeks to investigate the effects of supplementation of rumen-protected choline (RPC) or vitamin E on blood serum protein fractions, plasma total thiol molecules (TTM), and plasma total antioxidant capacity (TAC). Cows were randomly assigned to one of the following treatments: I -no supplement (control), II -90 g/d of RPC, and III -4400 IU/d of vitamin E. Serum protein electrophoresis of samples exhibited four main fractions in the blood serum of the cows including: albumin, α, β, and γ. The electrophoresis was carried out by capillary zone electrophoresis (CZE). In this study, feeding RPC or vitamin E affected the blood serum albumin fraction as well as blood plasma TTM (P<0.05) but the treatments did not affect the different fractions of globulin as well as plasma TAC (P>0.05). The results showed that the increases in serum albumin fractions and TTM which observed in this study, pointed towards a beneficial role of RPC and vitamin E in early lactating dairy cows.

Introduction-The neonatal period represents a critical stage in development of physiological functions. During this phase morphological, physiological and behavioral changes take place in newborn animals to permit a successful adaptation to the extra-uterine environment. Moreover, newborn lambs have limited energy reserves and need a rapid access to colostrum to maintain homeothermy and survive. In addition to energy, colostrum provides proteins, including immunoglobulins, which ensure passive systemic immunity. Therefore, getting early access to the udder is essential for the neonate. Aim-The purpose of this study was to monitor rectal temperature and the course of some metabolic parameters (serum total proteins, albumin, total cholesterol, triglycerides, creatinine and urea) of ten newborn lambs (Comisana breed) during the first 30 days of life in order to obtain useful information for neonatal care. Material and methods-For each animal rectal temperature was recorded using a digital thermometer (HI92704, Hanna Instruments Bedfordshire, UK) with the probe being inserted to a depth of 9 cm. From all animals blood samples were collected by jugular venipuncture at the same hour (9:00) every 3 days for 30 days starting from birth. By means of an UV Spectrophotometer, the serum concentration of total proteins, albumin, total cholesterol, triglycerides, creatinine and urea were determined. One-way repeated measures analysis of variance (ANOVA), followed by Bonferroni's Test, was used to determine statistically significant differences. Results and discussion-Data analysis of variance showed a statistical effect of days of life on total proteins (P<0.0003), albumin, total cholesterol, triglycerides (P<0.0001) and urea (P=0.0002), during the first 30 days of life, while no statistical significant effect of days of life on creatinine (P=0.09) and rectal temperature (P=0.06) is showed during the experimental period. Conclusion-The results obtained in this study make a contribution to the knowledge of physiological and metabolic modification in lambs during the first 30 days of life and give useful information for the diagnosis and treatment of neonatal diseases.

Background Saccharomyces cerevisiae (Baker&#39;s yeast) is found in diverse ecological niches and is characterized by high adaptive potential under challenging environments. In spite of recent advances on the study of yeast genome diversity, little is known about the underlying gene expression plasticity. In order to shed new light onto this biological question, we have compared transcriptome profiles of five environmental isolates, clinical and laboratorial strains at different time points of fermentation in synthetic must medium, during exponential and stationary growth phases. Results Our data unveiled diversity in both intensity and timing of gene expression. Genes involved in glucose metabolism and in the stress response elicited during fermentation were among the most variable. This gene expression diversity increased at the onset of stationary phase (diauxic shift). Environmental isolates showed lower average transcript abundance of genes involved in the stress response, assi...

Nitrogen is a crucial macronutrient needed in the greatest amount of all mineral elements required by plants. Development of crop varieties with high nitrogen use efficiency (NUE) is imperative for sustainable agriculture. Understanding how plant genes respond to different nitrogen conditions is essential for formulating approaches, for manipulating genes, for improving NUE. In the present study we analyzed the activity of three different enzymes involved in nitrogen assimilation viz., GS, GOGAT and GDH along with physiological parameters like chlorophyll variable yield (Fv/Fmax), photosynthesis rate and total chlorophyll content at four different growth stages of wheat plant development under different nitrogen treatments. For this study two different wheat varieties UP-2644 and Raj-4097 having high and low protein content, respectively in the grains were chosen. Gene expression profile of a Dof transcription factor (TaDof1 of wheat) was also included in the study to assess its role in nitrogen metabolism. Densitometry analysis at S 2 and S 3 stage of wheat spikes of both the wheat varieties grown at different nitrogen treatments showed that TaDof1 expression was up-regulated in low nitrogen treatment. In S 3 stage, in high protein content wheat variety UP-2644, TaDof1 expression was elevated in low and normal nitrogen treatment as compared to high nitrogen treatment. The gene expression profile of Dof 1 was found to coincide with the enzyme activities of GS, GOGAT at the S 3 stage. The activities of these enzymes were prolonged in the high protein content variety. Since, Dof transcription factor(s) have been previously reported to control the expression of genes involved nitrogen assimilation i.e., GS and GOGAT and may be the elevated expression of Dof 1 at the grain filling stage over expresses the GS and GOGAT genes thereby prolonging their activities.

MPG1, a pathogenicity gene of the rice blast fungus Magnaporthe grisea, is expressed during pathogenesis and in axenic culture during nitrogen or glucose limitation. We initiated a search for regulatory mutations that would impair nitrogen metabolism, M P G l gene expression, and pathogenicity. First, we developed a pair of laboratory strains that were highly fertile and pathogenic toward barley. Using a combinatorial genetic screen, we identified mutants that failed to utilize a wide range of nitrogen sources (e.g., nitrate or amino acids) and then tested the effect of these mutations on pathogenicity. We identified five mutants and designated them Nr-(for_nitrogenIegulation defective). We show that two of these mutations define two genes, designated NPRl and NPRP (for fiitrogen pathogenicity regulation), that are essential for pathogenicity and the utilization of many nitrogen sources. These genes are nonallelic to the major nitrogen regulatory gene in M. grisea and are required for expression of the pathogenicity gene MPG1. We propose that NPRl and NPRP are major regulators of pathogenicity in M. grisea and may be nove1 regulators of nitrogen metabolism in fungi.

Transgenic lines carrying a nodule-specific promoter fused to pat.: WdA were resistant to PPT only when nodulated. In some of these lines, nodule GS activity was completely resistant to soil applications of PPT under conditions where root GS was 100% inhibited. After long-term PPT treatment, one line (12E) showed a twofold increase in nitrogenase activity, a four-fold increase in GS activity, and a 50% increase in dry matter production. Possible explanations for how specific inhibition of root GS led to these effects are discussed.