Effects of nitrate and nitrite on dissimilatory iron reduction by Shewanella putrefaciens 200.

The inhibitory effects of nitrate (NO3-) and nitrite (NO2-) on dissimilatory iron (FE3+) reduction were examined in a series of electron acceptor competition experiments using Shewanella putrefaciens 200 as a model iron-reducing microorganism. S. putrefaciens 200 was found to express low-rate nitrate reductase, nitrite reductase, and ferrireductase activity after growth under highly aerobic conditions and greatly elevated rates of each reductase activity after growth under microaerobic conditions. The effects of NO3- and NO2- on the Fe3+ reduction activity of both aerobically and microaerobically grown cells appeared to follow a consistent pattern; in the presence of Fe3+ and either NO3- or NO2-, dissimilatory Fe3+ and nitrogen oxide reduction occurred simultaneously. Nitrogen oxide reduction was not affected by the presence of Fe3+, suggesting that S. putrefaciens 200 expressed a set of at least three physiologically distinct terminal reductases that served as electron donors to NO3-, NO2-, and Fe3+. However, Fe3+ reduction was partially inhibited by the presence of either NO3- or NO2-. An in situ ferrozine assay was used to distinguish the biological and chemical components of the observed inhibitory effects. Rate data indicated that neither NO3- nor NO2- acted as a chemical oxidant of bacterially produced Fe2+. In addition, the decrease in Fe3+ reduction activity observed in the presence of both NO3- and NO2- was identical to the decrease observed in the presence of NO2- alone. These results suggest that bacterially produced NO2- is responsible for inhibiting electron transport to Fe3+.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Growth and nitrogen metabolism of Catasetum fimbriatum (orchidaceae) grown with different nitrogen sources

Catasetum fimbriatum is an epiphytic orchid from South America that has been used for 15 years as a model plant for metabolic and developmental studies in our laboratory. In this work, C. fimbriatum plants were aseptically grown with 6 mol m(-3) of either glutamine or inorganic nitrogen forms (NO(3)(-):NH(4)(+) ratios). The highest biomass accumulation was found in plants supplied with glutamine; no significant difference was observed in plants incubated in the presence of inorganic nitrogen sources. Nitrogen assimilation was limited in the presence NO(3)(-) as a sole nitrogen source. C. fimbriatum did not accumulate NO(3)(-) and very low rates of in vivo nitrate reductase activity were observed. Most nitrate reductase activity (70%) was detected in the 2 cm apical roots. Nitrate-treated plants exhibited relatively lower amounts of free amino-N, chlorophyll and free NH(4)(+) contents and higher soluble sugar contents than the NH(4)(+)-treated plants. While shoot glutamine synthetase activity was only slightly affected by nitrogen sources, root glutamine synthetase activity was not modified by any nitrogen form. Glutamate dehydrogenase-NADH activity in shoot tissues was not influenced by any nitrogen source. However, the glutamate dehydrogenase-NADH activity in roots was enhanced when NH(4)(+) tissue contents was augmented by increasing NH(4)(+) in the medium and by the presence of glutamine. Our results strongly suggest that organic nitrogen and NH(4)(+) are probably the most important nitrogen sources to C. fimbriatum plants.     

Influence of Different Concentrations of NO-3 and NH+4 on the Activity of Glutamine Synthetase and Other Relevant Enzymes of Nitrogen Metabolism in Wheat Roots

Influence of different concentrations of NO3 and NH+ on the activity of glutamine synthetase (GS), asparagine synthetase (AS), glutamate dehydrogenase (GDH), nitrate reductase (NR) and the changes of GS-mRNA in wheat roots have been studied with enzymes activity assay and Northern blot. The results showed that the higher GS activity was found in roots of wheat when NH+4-N was the sole nitrogen source than when NO3-N was the sole nitrogen source. GS-mRNA of Northern blot was simillar to GS activity. 3 mmol/L NO3- promoted the activity of AS. The change of AS was independent of the change of GS. GDH activity was not been detected, and change in regulation of NR activity was not found.     

Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia

Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO₃⁻-inducible. NH₄⁺ was the initial repressor signal for the uptake process which was involved in the control of the NO₃⁻inducible reductase system.     

Effect of applied benzyladenine on endogenous cytokinin content during the early stages of bud development of kiwifruit

The filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium-grown cultures of the irreversible inhibitor of glutamine synthetase activity L-methionine-D, L-sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re-established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX-treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.     

外源Spd对盐碱胁迫下番茄幼苗氮代谢及主要矿质元素含量的影响

采用水培方法,研究了盐碱与Spd处理对两品种番茄（中杂9号和金棚朝冠）幼苗氮代谢及主要矿质元素含量的影响.结果表明： 盐碱胁迫下,番茄幼苗干生物量显著减少,植株生长受到抑制;叶片和根系硝酸还原酶（NR）、谷氨酰胺合成酶（GS）、谷氨酸合成酶（GOGAT）活性及硝态氮（NO₃^--N）、全N、全K、Ca²⁺、Mg²⁺含量显著降低,铵态氮（NH₄⁺-N）、Na⁺含量显著增加;两品种叶片及中杂9号根系谷氨酸脱氢酶（GDH）活性显著升高,金棚朝冠根系GDH活性变化不显著;叶片全P含量显著降低,根系全P含量显著升高（金棚朝冠）或无显著变化（中杂9号）.Spd处理通过增强NR、GS、GOGAT活性提高了植株对NH₄⁺的同化利用率,有效缓解了盐碱胁迫导致的氮代谢紊乱,进而促进不同器官对P、K、Ca、Mg、Na的吸收、释放或转运,在一定程度上维持了各元素之间的相对平衡,从而增强植株对逆境的适应能力.此外,盐碱对中杂9号的抑制作用及外源Spd对其氮代谢紊乱和营养失衡的缓解作用高于金棚朝冠.     

Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+.

Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii.     

Light mediated regulation of nitrate assimilation in Synechococcus leopoliensis

Synechococcus leopoliensis was cultivated in a light/dark regime of 12:12 h. After onset of the illumination (2 h), the specific activity of nitrite reductase, glutamine synthetase and isocitric dehydrogenase increased; that of glucose-6-phosphate dehydrogenase decreased and that of nitrate reductase and NAD- (NADP) glutamate dehydrogenase remained nearly unchanged.This stimulation of the enzymes in vivo was also observed in vitro. Also, when extracts from darkened cells were incubated with thioredoxin and dithioerythriol enzyme activities increased in the same amount as obtained in vivo. In addition, glucose-6-phosphate dehydrogenase and isocitric dehydrogenase were stimulated by Mn²⁺ and Mg²⁺ in the assay mixture. Glutamine synthetase activity was enhanced only by Mg²⁺ while Mn²⁺ was inhibitory.The results are discussed with respect to the regulation of nitrogen metabolism by light.Abbreviations GS glutamine synthetase - GOGAT glutamate-oxoglutarate-aminotransferase - TR thioredoxin - DTE dithioerythritol - LD change from light to dark     

盐胁迫下外源NO对苜蓿幼苗生长及氮代谢的影响

为探寻增强苜蓿耐盐能力的调控途径,以甘农4号苜蓿品种为材料,采用NO供体硝普钠、NO清除剂c-PTIO及硝普钠类似物亚铁氰化钠处理苜蓿幼苗,研究盐胁迫下外源NO对苜蓿幼苗生长、光合特征、氮同化酶活性和氮代谢物含量的影响.结果表明: 外源NO能明显缓解盐胁迫对苜蓿幼苗生长及光合作用的抑制,单株干质量、叶绿素含量、净光合速率、蒸腾速率和可溶性蛋白含量增加;外源NO能增强硝酸还原酶、谷氨酰胺合成酶和谷氨酸合酶活性,抑制蛋白水解酶和谷氨酸脱氢酶活性, 降低叶片中游离氨基酸含量,提高硝态氮含量,加快铵的同化.NO供体SNP的类似物亚铁氰化钠对盐胁迫下苜蓿幼苗生长及氮代谢无调控作用;施用NO清除剂c-PTIO加剧了盐胁迫对苜蓿幼苗生长和氮代谢的抑制,添加外源NO能缓解c-PTIO的抑制效应.盐胁迫下,外源NO和内源NO均参与了苜蓿幼苗氮代谢的调控.     

A comparison of the influence of iron on the growth and nitrate metabolism of Anabaena and Scenedesmus

A comparative study of growth and nitrate metabolism of Anabaena flos-aquae (Lyng.) Bréb. and Scenedesmus bijugatus var. seriatus Chodat investigated possible mechanisms for the iron-stimulated increases in growth specific for blue-green algae in mixed algal communities. Algae were separately grown in an morganic medium with varying concentrations of iron and nitrate to determine the effects on each organism. Iron was found to be a limiting nutrient for cultures of both Anabaena and Scenedesmus as determined by chlorophyll a concentrations and cell enumeration. Both iron and nitrate stimulated the specific activity of nitrate reductase, nitrite reductase, and glutamine synthetase in Anabaena. Iron enrichment did not increase the activity of the enzymes in Scenedesmus, but inhibited the activity of nitrate reductase and glutamine synthetase. The stimulation of growth by iron in cells grown under iron limiting conditions was associated with increased nitrate metabolism in Anabaena but not in Scenedesmus.     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Nitrate reductase and glutamine synthetase activities,nitrate and ammonia assimilation,in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

金属离子对粪产碱杆菌C16的脱氮和亚硝酸盐积累的影响

【目的】研究不同金属离子对异养氨氧化细菌C16的生长和脱氮性能影响,探讨适于C16生长和脱氮的金属离子及其浓度。【方法】实验选用Mg2+、Mn2+、Fe2+、Cu2+、Zn2+5种金属离子,对C16的生长﹑脱氮性能﹑亚硝酸盐氮积累以及相关酶活性进行研究。【结果】Mg2+明显促进C16的生长和NH4+-N氧化速率;较高浓度Mn2+使得C16无法生长;原培养基中缺少Fe2+会抑制C16的生长和NH4+-N氧化速率;在原培养基中加入0.1 mmol/L的Cu2+对C16的生长和脱氮具有一定的促进作用,Cu2+使得培养基中基本无NO2--N和NH2OH的积累;不同浓度的Zn2+对C16的生长和氨氮去除有抑制作用。酶活实验结果显示,0.1 mmol/L Mg2+促进了羟胺氧化还原酶(HAO)的活性;0.1 mmol/L Cu2+促进了硝酸盐还原酶(Nar)和亚硝酸盐还原酶(Nir)的活性。【结论】Mg2+是C16生长和脱氮过程中的一种重要金属离子;加入Cu2+可避免过量亚硝酸盐积累。     

Repression of nitrate reductase in Neurospora studied by using L-methionine-DL-sulfoximine and glutamine auxotroph gln-1b.

The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-1b, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.     

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

Nitric oxide plays a role as second messenger in the ultraviolet-B irradiated green alga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

Nitric oxide (NO) stimulated the activity of plasma membrane H⁺-ATPase, 5′-nucleotidase, peroxidase, ascorbate peroxidase and glutathione reductase in ultraviolet B (UV-B) irradiated Chlorella pyrenoidosa. It also boosted the activity of nitrogen-metabolism enzymes such as nitrate reductase, nitrite reductase, glutamine synthetase, which were inhibited by UV-B irradiation. The chlorophyll fluorescence ratio (Fv/Fm) of the UV-B irradiated algae and decreased continuously after the cells were transferred to UV-B irradiation. A continuing decrease of the Fv/Fm was observed even after the cells were transferred to photosynthetically active radiation (PAR). After adaptation for 8 h under PAR (after treatment with nitric oxide), Fv/Fm recovered to 55 % of normal levels — without NO the value approached zero. Exogenous NO stopped the decay of chlorophyll and thylakoid membrane in cells exposed to UV-B irradiation. NO plays probably a key role in damage induced by UV-B irradiation in green algae.