Search

Despite being an autotrophic organism, Galdieria phlegrea (Galdieriaceae) has the ability to use glycerol thereby switching to heterotrophy in the dark and mixotrophy in the presence of light. To examine cellular changes during the switch to mixotrophic metabolism and finally to photoautotrophic metabolism, heterotrophic cells of G. phlegrea were exposed to light and split into two subcultures. Cells exposed only to light but cultivated in medium containing glycerol grew with a recovery time of at least 3 days. In a parallel culture, the simultaneous removal of glycerol from the culture medium and light exposure allowed Galdieria cells to rapidly recover their growth rate due to their ability to rapidly absorb ammonium from the medium. However, contrary to expectation, a higher content of total soluble protein was observed in light-exposed cells cultivated in medium containing glycerol compared to cells cultivated without glycerol. In addition, the level of Rubisco in cells exposed to light and cultivated without glycerol was higher than those in cells cultivated in medium containing glycerol, indicating full photosynthetic functionality after only 3 days of light treatment. The greater chlorophyll a content confirms that the photosynthetic activity of cells cultivated without glycerol recovers earlier than that of heterotrophic cells exposed to light but still cultivated in medium containing glycerol.

In plants and algae, sulfate assimilation and cysteine synthesis are regulated by sulfur accessibility from the environment. This study reports the effects of sulfur (S)-deprivation in autotrophic and heterotrophic cultures of Galdieria phlegrea (Cyanidiophyceae), a unicellular red alga isolated in the Solfatara crater located in Campi Flegrei (Naples, Italy), where H2S is the prevalent form of gaseous sulfur in the fumarolic fluids and sulfur is widespread in the soils near the fumaroles. This is the first report on the effects of S-deprivation on a sulfurous microalga that is also able to grow heterotrophically in the dark. The removal of sulfur from the culture medium of illuminated cells caused a decrease in the soluble protein content and a significant decrease in the intracellular levels of glutathione. Cells from heterotrophic cultures of Galdieria phlegrea exhibited high levels of internal proteins and glutathione content, which did not diminish during S-starvation, but rather glutathione significantly increased.The activity of O-acetylserine(thiol)lyase (OASTL), the enzyme synthesizing cysteine, was enhanced under S-deprivation in a time-dependent manner in autotrophic but not in heterotrophic cells. Analysis of the transcript abundance of the OASTL gene supports the OASTL activity increase in autotrophic cultures under S-deprivation.

To date, the main source for phycocyanin production is the thermophilic cyanobacterium Spirulina platensis, although latest researches are exploring the possibility to exploit the thermoacidophilic Cyanidiophyceae (Rhodophyta) to this purpose. Galdieria phlegrea is a polyextremophilic red alga belonging to Cyanidiophyceae, colonizing, along with G. sulphuraria, acidic and thermal environments and distinguishable from this latter on the base of molecular and ecophysiological traits. In the present paper, a characterization of C-phycocyanin from two strains of G. phlegrea with different geographic provenance (Phlegrean Fields Naples, Italy; Diyadin, Turkey) was provided under autotrophic and heterotrophic conditions. The results showed different optimal pHs and thermostability between the strains and between autotrophic and heterotrophic cells. What is intriguing, a pre-heating at 70 °C of heterotrophic cells from the Italian strain resulted in a highly heat-resistant of C-phycocyanin. We hypothesized that the different features of this pigment, currently widely used for various applicative purposes, are related to the habitat from which the microalga comes from.

Effect of temperatures and illumination of temperate winter on photosynthesis and respiration was studied in the psychrophilic microalgae, Koliella antarctica (Trebouxiophyceae). Outdoor and indoor algal cultures were compared. Photosynthetic as well as respiration rates increased as light and temperature increased, until 35 °C, more in outdoor than in indoor cells, in agreement with the calculated Q10 values. K. antarctica showed important strategy mechanisms of adaption to the several temperature and light conditions. These significant photo-acclimation and thermo-acclimation abilities make it possible to cultivate Koliella for different uses, under less expensive outdoor conditions. Therefore, K. antarctica shows important strategy mechanisms of adaption to various temperature and light conditions; moreover, by varying the culture conditions, it is possible to modulate and optimize the growth and accordingly the biomass production. This is a very interesting point since it has been proved that this microalga is a promising potential source of functional ingredients, such as polyunsaturated fatty acids and carotenoids, suitable for industrial purposes.

Effect of temperatures and illumination of temperate winter on photosynthesis and respiration was studied in the psychrophilic microalgae, Koliella antarctica (Trebouxiophyceae). Outdoor and indoor algal cultures were compared. Photosynthetic as well as respiration rates increased as light and temperature increased, until 35 °C, more in outdoor than in indoor cells, in agreement with the calculated Q(10) values. K. antarctica showed important strategy mechanisms of adaption to the several temperature and light conditions. These significant photo-acclimation and thermo-acclimation abilities make it possible to cultivate Koliella for different uses, under less expensive outdoor conditions. Therefore, K. antarctica shows important strategy mechanisms of adaption to various temperature and light conditions; moreover, by varying the culture conditions, it is possible to modulate and optimize the growth and accordingly the biomass production. This is a very interesting point since it has been proved that this microalga is a promising potential source of functional ingredients, such as polyunsaturated fatty acids and carotenoids, suitable for industrial purposes.

Nitrogen (N) and sulphur (S), being essential macronutrients, have important roles in microalgae metabolism. Effects of N- or S-shortage were investigated in the green microalgae Chlorella sorokiniana subjected to 24 h of starvation, by measuring the glutamine synthetase (GS) and O-ace- tylserine(thiol)lyase (OASTL) activities, proteins and amino acids levels. To test possible metabolic impact related to carbon (C) metabolism in response to N- or S-deprivation, starch and total C, N and S contents were also determined. The growth of C. sorokiniana cells was affected by N or S availability. The algae cultured for 24 h in a medium deprived of nitrogen or sulphur showed a decrease in the growth rate and changes in the average volume cell. Nitrogen starvation affected proteins level in the algae cells more than S-deprivation did. The decline in the protein levels observed under S-deficient conditions was coupled with the accumulation of the amide glutamine and with OASTL activity increase; additionally, N-deficiency promoted a decrease in cysteine (Cys) levels (50%) and an increase in GS activity. Nevertheless, S-deprivation had negligible effects on GS activity, while N-deprivation significantly affected OASTL activity. Total C was also estimated in cells N- or S-deprived; nitrogen deprivation strongly affected total C content more than S-deprivation, which in addition reduced the content of C and N, but leaves intact their ratios. Our results support the hypothesis that in Chlorella sorokiniana cells a reciprocal influence of N, S and C assimilation occurs.

Summary • Temperature effects on growth, photosynthesis, respiration and nitrate reductase (NR) were studied in the cryophilic algae Koliella antarctica and ‘Chlorella’saccharophila, and in the mesophilic Chlorella sorokiniana. • Growth rate was measured as increase in optical density. Photosynthesis at saturating light and respiration in darkness were measured as O2 exchange. NADH : NR was assayed in crude extracts. • The two cryophilic algae grew below 15°C, and C. sorokiniana above 20°C. Photosynthetic and respiration rates of K. antarctica and ‘C.’ saccharophila were elevated at 5°C, and peaked at 30°C. Arrhenius plots from 5 to 25°C were linear in K. antarctica, whereas in ‘C.’ saccharophila and C. sorokiniana they exhibited breaks at 15 and 20°C, respectively. Values for activation energy (Ea) and the factor by which the rate increases with raising the temperature 10°C (Q10) differed. Nitrate reductase had its optimum at 25°C in cryophilic algae and at 35°C in C. sorokiniana. • We conclude that growth of cryophilic algae at low temperature is favoured by elevated photosynthesis and respiration rates, but that it could be limited by a high respiration : photosynthesis ratio.

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP+/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, Km for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.

N-limited cells of Chlorella sorokiniana Shihira and Krauss 211/8k exhibited rates of respiratory oxygen consumption and photosynthetic oxygen evolution which were 70 and 17%, respectively, of those of N-sufficient cells. N-sufficient cells absorbed NH 4 + in light at a linear rate, and absorption was 100% inhibited by darkness. N-limited cells, by contrast, absorbed NH 4 + at almost similar rates in light and darkness. The rate of uptake in darkness decreased after 20 min, and decreased further after 60 min. In the light, by contrast, it decreased to a constant higher value, which appeared to approximate the sum of the rate of the dark absorption plus the rate of the light-dependent NH 4 + absorption by N-sufficient cells. NH 4 + in darkness did not affect respiration of N-sufficient cells. However, NH 4 + supply to N-limited cells in darkness resulted in a sudden increase of respiratory oxygen consumption, suggesting that short-term control mechanisms were triggered; it also resulted in time-dependent increase in photosynthetic activity and chlorophyll content, suggesting long term control of the photosynthetic apparatus. NH 4 + supply also resulted in a resumption of growth, in light but not in darkness. It is proposed that the use of NH 4 + in darkness by N-limited cells serves to recover from N-limitation, whereas in light it serves to sustain both recovery and growth. This could also explain why cells which are in an N-sufficient status and need nitrogen for growth only, exhibited exclusively a light-dependent NH 4 + uptake.

The heavy metal pollution in soils and aquatic environments is a serious ecological problem. In the green-microalga Chlorella sorokiniana 211-8K (Chlorophyceae) exposed to ions of lead (Pb) and cadmium (Cd) we studied the metabolic responses to the toxicity of these two heavy metals. Our data indicate that both the pollutants alter the alga cell ultrastructure and its physiological characteristics (growth, photosynthesis, respiration, enzyme activities). The toxic effects of the two metals resulted time-dependent to the exposure. After 24 h of treatment with 250 μM Pb or Cd, photosynthesis was inhibited until to 77 and 86%, however respiration was strongly enhanced up to 300 and 350%, respectively. In the algal cells Pb or Cd exposure induced a reduction in the content of the total chlorophylls and a decrease of the soluble protein levels, significantly compromising the growth, particularly in cultures cadmium-treated. We report data on ultrastructural changes induced by the two heavy metals; they affected overall chloroplast ultrastructure of the alga. Most importantly, the O-acetyl-L-serine(thiol)lyase (OASTL) activity was appreciably increased after only 2 h of Cd exposure, indicating the existence of a link between the metal contamination and cysteine synthesis. Then, Chlorella sorokiniana cells seem to better tolerate high concentrations of Pb while appear to be more sensitive to Cd ions. These results provide some additional information that can lead to better understand consequences of heavy metal poisoning in microalgae.

We analyzed the effects of deprivation and subsequent restoration of sulphate (S) in the nutrient solution on cysteine (Cys) and O-acetyl-L-serine (OAS) levels in Chlorella sorokiniana (211/8k). The removal of S from the culture medium caused a time-dependent increase in O-acetyl-L-serine(thiol)lyase (OASTL) activity and a decrease in soluble proteins content. The protein gel blot analysis was used to show that OASTL isoforms are located in the chloroplast and in the cytoplasm of S-starved cells. S-deprivation caused a decrease in the intracellular levels of Cys and glutathione (GSH) and an increase in serine (Ser) and OAS, reflecting an imbalance between sulphur and nitrogen assimilation. Re-supplying of sulphate to S-starved cells produced a decrease in OAS levels and concomitant rapid increase in Cys and GSH concentrations. The simultaneous addition of OAS and sulphate to S-starved cells did not further increase the concentration of Cys, suggesting the existence of a threshold level of intracellular Cys that is independent of the cellular concentration of OAS. Our findings that OAS is stored during S-starvation and that its quick decrease appears to be coupled with the increase of Cys levels upon re-supply of sulphate, imply that the central role that these two compounds play is in the regulation of sulphur-assimilating enzymes in response to the S status of the cell.

The mixed effects of nitrogen nutrition and sulphate assimilation were investigated in barley plants (Hordeum vulgare var. Alfeo) that were subjected to long-term sulphur and/or nitrogen starvation, by measuring the O-acetylserine(thio)lyase (OASTL–EC 4.2.99.8) activity, changes in –SH compounds and amino acid levels. The growth of barley plants cultured in the hydroponic vessels was severely affected by altered nutrient levels. The barley plants grown in medium deprived of nitrogen and/or sulphur sources for 21 days showed increase in both root length and weight. In contrast, the shoot growth was reduced in nitrogen-starved plants and was unaffected by sulphur deprivation. Sulphur starvation affected the level of proteins in barley plants more than nitrogen deprivation. The decline in the protein levels observed under sulphur-deficient conditions was coupled with the accumulation of glutamine, asparagine and serine, mainly in the roots; additionally, a nitrogen deficiency in the roots promoted a decrease in both glutathione and cysteine levels. The simultaneous deprivation of nitrogen and sulphur in plants leads to an alteration in their metabolism; high levels of glutathione (GSH) in the shoots could signify the induction of a mechanism intended for coping with stressful conditions. Sulphate deprivation enhanced OASTL activity, mainly in the roots; on the other hand, OASTL increases observed under S deprivation were clearly dependent on the nitrogen availability in the culture medium. In fact, the nitrate supply to the N and S starved plants that showed OASTL activity very low, rapidly recovered the OASTL activities to the levels typical of control plants. Nevertheless, the ammonium supply had negligible effects on the OASTL activity only observed after three days in the roots. Our results support the hypothesis that in barley plants, a portion of S assimilation (up to cysteine biosynthesis) occurs in the roots, and a reciprocal influence of nitrogen assimilation on cysteine synthesis occurs.

In N-sufficient cells of Chlorella sorokiniana Shihira and Krauss strain 211/8K (CCAP of Cambridge University), assimilation of ammonium was strictly dependent on light and CO 2 , and was severely inhibited by 100 μM atrazine or 10 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In N-limited cells, assimilation of NH 4 + took place at similar rates in both light and darkness, which were 1.6-fold higher than the rate of light-dependent assimilation by N-sufficient cells. Assimilation by N-limited cells was inhibited by L-methionine-DL-sulfoximine (MSX), but not by atrazine or DCMU

Unicellular algae represent an interesting source of bioactive food ingredients. In this study, fatty acids and carotenoids of Koliella antarctica, a psychrophylic Antarctic unicellular alga, were investigated. K. antarctica was cultivated at different temperatures and harvested at the early exponential growth phase and at the late exponential growth phase. After 240 h, at the end of the late exponential growth phase, the cell biomass concentration was 1.1 g L− 1 at 10 °C while it was 2.2 g L− 1 at 15 °C. The lipid profile of K. antarctica was rich in polyunsaturated fatty acids (PUFA) particularly at the early exponential phase (50.8% of total fatty acids). The comparison of data obtained from algae cultivated at 10 °C and 15 °C, showed at 15 °C a strong increase in saturated lipids and a decrease in PUFA. At the same time the presence of EPA and DHA was marginally affected, with a percentage decrease around 15–20%. The productivity of EPA and DHA was similar at the two growth temperatures with maximum values of 0.6 mg L− 1 day− 1 and 1.3 mg L− 1 day− 1, at 10 and 15 °C, respectively. In K. antarctica cultures, the concentration of lutein and astaxanthin increased during growth up to 1.4 g per 100 g dry weight for both compounds. This is one of the highest concentrations reported in the literature for carotenoid production by unicellular algae, with a total carotenoid productivity of 3.1 mg L− 1 day− 1. Although optimization studies to increase K. antarctica biomass production were not yet carried out, data suggested that this alga is a promising source for low cost production of valuable bioactive compounds. The performance recorded at 15 °C suggested that it could be particularly suitable for outdoor cultivation during the winter season in the Mediterranean region.

Heavy metals enter ecosystems by both natural and anthropogenic processes. An excess of metals in the environment damages plant metabolism, leading to oxidative stress in the cell. Most metal ions present in the cell are bound to low molecular mass ligands. The major types of S-containing ligands involved in metal binding are derived from cysteine. The authors show, for the first time in Scorpiurum circinatum, a Mediterranean epiphytic moss, that cysteine synthesis is linked to environmental heavy metal pollution. In moss bag exposed in strongly heavy metals polluted sites, O-acetylserine(thio)lyase (OASTL) activity seems strongly related to heavy metal exposure. In addition, heavy metal exposure in S. circinatum caused ultrastructural alterations of vacuolar system and thylakoid organisation, as shown through Transmission Electron Microscopy (TEM) observations. Because S. circinatum is able to tolerate and accumulate various heavy metals, the authors suggest the OASTL enzymatic assay in S. circinatum as biomarker of heavy metals.

The possible origin of carbon skeletons for ammonium assimilation in Cyanidium caldarium (Tilden) Geitler was investigated. N-sufficient cells assimilated ammonium at a rate of 182 ± 18 μmol·mL packed cell volume (pcv)-1· h-1. Removal of CO2 or darkening almost immediately prevented ammonium assimilation. N-limited cells in light assimilated ammonium at a rate of 493 ± 45 μmol · mL pcv-1· h-1 in the presence of CO2 and at a lower rate of 168 ± 17 μmol · mL pcv-1· h-1 in the absence of CO2. In darkness they assimilated ammonium at a rate of 293 ± 29 μmol · mL pcv-1 h-1 in the presence of CO2, only 60% of the assimilation rate in light. In the absence of CO2, ammonium was assimilated at a similar rate of 325 ± 14 μmol · mL pcv-1· h-1. Under the latter conditions, however, assimilation was inhibited after 40 min and ceased after 70 min; it resumed upon resupply of CO2. We suggest that N-sufficient cells of C. caldarium obtain carbon skeletons for ammonium assimilation exclusively by photosynthetic reactions. Upon N-limitation they develop the ability, apparently through derepression or activation of regulatory enzyme system(s), to obtain a consistent quantity of additional carbon skeletons and ATP from mobilization of carbon reserves. This enables the N-limited cell to assimilate ammonium not only in light but also in darkness, and at a higher rate than N-sufficient cells. The fact that ammonium assimilation in light occurs at a higher rate than in darkness suggests that ammonium assimilation in light is the sum of both light and dark ammonium assimilation, which implies separate metabolic reactions for the two processes. These results suggest the existence of two distinct and differently controlled pathways in N-limited cells, but not in N-sufficient cells, through which carbon skeletons for ammonium assimilation originate. An important role for dark CO2 fixation in dark or light ammonium assimilation is also indicated.

Cells of Chlorella sorokiniana Shihira & Krauss 211/8k, grown in a chemostat under conditions of nitrate limitation, assimilated ammonium in light (307 ± 12 μmol · mL packed cell volume (pcv)−1· h−1) and darkness (305 ± 30 μmol · mL pcv−1· h−1) at similar rates when supplied with air containing 5% CO2. Ammonium assimilation in cell suspensions flushed with pure nitrogen to establish anaerobic conditions was almost totally prevented both in darkness and in light. Cell suspensions flushed with CO2−-free air and grown in the dark assimilated ammonium at a rate of 314 ± 23 μmol · mL pcv−1· h−1. Light assimilation was 128 ± 10 μmol · mL pcv−1· h−1, a rate which was only 41% of that in darkness. Resupply of ammonium to N-limited chemostat cells resulted in a 3.5-fold stimulation of dark respiration and a suppression of photosynthetic O2 evolution. We hypothesize that the partial decline of ammonium assimilation in Chlorella 211/8k in the light under conditions of limited CO2 supply is due to the fact that the photosynthetic carboxylation reaction competes with the dark carboxylation reaction for the available CO2, and that the production of C4 and C5 precursors foramina acid biosynthesis will be slower than in the dark. Alternatively we suggest that an increase in the concentration of photogenerated compound(s) not used for CO2 assimilation attains an intracellular level suitable to trigger a control mechanism operating at the level of starch break-down or glycolysis.

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35°C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10°C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45°C, respectively, with only 33 and 23% of maximal activities being retained at 10°C. In presence of 2 μ m flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35°C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25°C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10°C (Q10) and apparent activation energy (Ea) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10°C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure. Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35°C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10°C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45°C, respectively, with only 33 and 23% of maximal activities being retained at 10°C. In presence of 2 μ m flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35°C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25°C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10°C (Q10) and apparent activation energy (Ea) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10°C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure. © 2006 Blackwell Publishing Ltd.

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki(NADPH) is 103 microM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki(NADPH) decreased to 48 microM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.

In barley (Hordeum vulgare L. var. Nure), glutamate synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) are strictly correlated biochemical processes. NADHGOGAT was the major root isoform, whose activity increased on a medium supplied with NH 1 4 or NO 2 3 ;b y contrast, no noticeable variations could be observed in the leaves of plants supplied with nitrogen. In the leaves, the major isoform is Fd-GOGAT, whose activity increased under nitrogen feeding. G6PDH activity increased in the roots supplied with nitrogen; no variations were observed in the leaves. Moreover, an increase of the P2 isoform in the roots was measured, giving 13.6% G6PDH activity localized in the plastids under ammonium, and 25.2% under nitrate feeding conditions. Western blots confirmed that P2-G6PDH protein was induced in the roots by nitrogen. P1G6PDH protein was absent in the roots and increased in the leaves by nitrogen supply to the plants. The changes measured in cytosolic G6PDH seem correlated to more general cell growth processes, and do not appear to be directly involved in glutamate synthesis. The effects of light on Fd-GOGAT is discussed, together with the possibility for P2-G6PDH to sustain nitrogen assimilation upon illumination.

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose-6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots (Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine-fed barley roots showed a similar increase in G6PDH activities to ammonium-fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium-fed and glutamine-fed roots. The isozymes had different pH optima and apparent Km values for glucose-6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.

N-sufficient cells of Chlorella sorokiniana Shihira and Krauss, strain 211/8k, absorbed NH + 4 under light plus CO 2 conditions, when growth occurred, but not in darkness or in the absence of CO 2 , when growth was inhibited. N-sufficient cells subjected to conditions of N-starvation for a 24-h period showed a marked loss of photosynthetic activity. Upon supply of NH + 4 , N-starved cells sufflated with CO 2 air exhibited a time-dependent recovery of photosynthetic activity, both when suspended in light and in darkness. By contrast, growth only occurred in cells suspended in light. N-starved cells absorbed NH + 4 in darkness, but at a lower rate than in light. All of these data suggest that dark NH + 4 uptake is driven by N assimilation to recover from N-starvation and that the light-dependent NH + 4 uptake is driven by growth, being then influenced by conditions that affect recovery or growth. Unlike CO 2 conditions, in a CO 2 -free atmosphere, absorption of NH + 4 by N-starved cells occurred at a higher rate in darkness than in light. Accordingly, resumption of photosynthetic potential after NH + 4 supply occurred in darkened cells, but not in illuminated cells. Respiratory activity of N-starved cells was enhanced up to 3-fold by NH + 4 and 2-fold by methylammonium, with different patterns, suggesting that respiratory enzymes were affected by N-metabolism, especially through short-term control mechanisms triggered by the expenditure of metabolic energy involved in N-metabolism.

Barley plants (Hordeum vutgare L.) grown for 10 d in nitrogen-free hydroponic culture, after a rapid initial phase absorbed supplied NH4 (+) at a constant rate of 15.1 ±1.2 μ mol h(-1) g(-1) f. wt in the light, arid at a rate of 13.81 ± 1.6 μ mol h(-1) g(-1) f. wt in darkness. Ammonium-grown plants assimilated NH4 (+) at a rate of 7.5 ± 0.33 μmol h-1 g(-1) f. wt and at a 50% lower rate in darkness. Nitrogen-free grown plants showed low concentrations of free amino acids in both root and shoot tissues. Supplying NH4 (+) caused an immediate increase in the concentration of free amino in the root tissues of both illuminated and darkened plants over a 120 mm period. The increase in concentration of glutamine then exhibited a lag period of 120 min, after which it resumed, but to a very small extent. Glutamine also accumulated in shoot tissue of illuminated plants at increasing rates, attaining a concentration which, 8 h after NH4 (+) supply, was 1.61-fold greater than that attained in the roots. In shoots of darkened plants, by contrast, the concentration of glutamine increased slowly and was always smaller than that in the root tissue. Overall formation of glutamine (in shoots and roots) occurred at decreasing rates during the first 4 h, and then at increasing rates. The increase was more pronounced in illuminated plants than in darkened plants, liven 24 h after NH4 (+) was supplied, glutamine content in root tissue was lower than that in shoot tissue. However, 48 h later, the concentrations of glutamine in root and shoot were similar, attaining values that were almost 47-fold (in root) and 134-fold (in shoot) greater than initial values. Significant levels of asparagine were detected in the root and in the shoot 24 h after adding NH4 (+) . These increased further during the succeeding period. Ammonium supply caused a transitory drop in the concentration of ATP in root tissue, along with noticeable transitory variations in glucose-6-P concentration. A permanent decrease in free glucose concentration was also detected. Addition of NH4 (+) caused 2- and 1.43-fold increases in respiratory oxygen consumption by roots of illuminated and darkened plants, respectively. Both in the light and in the dark, the root tissue accumulated methylammonium up to a concentration of 55-67 μmol h(-1) g(-1) f. wt. Methylammonium was never found in shoot tissue of either illuminated or darkened plants. Methylammonium stimulated respiration of root barley plants by a factor of 1.2. Regulatory aspects of NH4 (+) metabolism are discussed.

Thermophilic eukaryotic algae are rare in acid hot environments in nature. The sole photosynthetic organisms living in thermal alkaline environments are almost exclusively cyanobacteria. Among algae, only the members of the class Cyanidiophyceae [including the families Cyanidiaceae and Galdieriaceae (Merola et al. 1981)] were found to live in thermal acidic habitats up to 57 °C (Doemel and Brock 1970). The family Cyanidiaceae includes the alga Cyanidium caldarium. For a larger spec trum on these algae, see chapters by Seckbach, Ott and by Sentsova in this volume.

Amino acid pools in Cyanidium caldarium (Tilden) Geitler, growing under conditions of sufficiency of nutrients (sufficient cells), or under conditions of nitrogen, potassium or phosphorus limitation (N-, K- or P-limited cells), and responses upon nutrient resupply were investigated. Glutamate was the dominant amino acid in sufficient cells [11.2 μmol ml −1 packed cell volume (pcv)], in N-limited cells (7.71 μmol ml −1 pcv) and in P-limited cells (7.46 μmol ml −1 pcv), whereas in K-limited cells the dominant amino acid was alanine (8.83 μmol ml −1 pcv). Other amino acids detected occurring at significantly high concentrations were: citrulline, alanine, aspartate, glutamine and δ-aminolevulinic acid in sufficient cells, alanine in N-limited cells; alanine, citrulline, serine, arginine and asparagine in P-limited cells; glutamate, arginine, glutamine, citrulline, asparagine, serine, aspartate and putrescine in K-limited cells. On adding NH 4 + to N-limited cells the level of glutamate decreased and then increased to the starting value; there occurred an increase in the level of glutamine, aspartate, citrulline, arginine, alanine, serine, as well as of δ-ALA and putrescine. Resupply of Pi to P-limited cells caused a significant decrease of glutamate and a concomitant increase of alanine, but was without efrect on the concentration of other amino acids. On adding K + to K-limited cells there was an increase of glutamate and citrulline, and a decrease of putrescine, whereas arginine and alanine remained unvaried at the very high level occurring before addition.

Growth, intracellular free amino acid pools and photosynthetic and respiratory activities in nutrient sufficient cells and in N-, K- and P-limited cells of Cyanidium caldarium (Tilden) Geitler, and responses to nutrient resupply were investigated. Addition of ammonium to N-limited cells and of phosphate to P-limited cells resulted in a stimulation of dark respiration and in a decrease in photosynthetic oxygen evolution. Addition of K to K-limited cells had no effect on rates of photosynthesis and respiration. Nutrient limited cells and sufficient cells exhibited different free amino acid profiles. Upon resupply of ammonium to N-limited cells levels of glutamine, citrulline, arginine, alanine, and serine increased. Also the levels of delta-aminolevulinic acid (delta-ALA) and putrescine increased notably. On adding phosphate to P-limited cells the level of glutamate decreased significantly whereas the level of alanine increased and the concentrations of other amino acids remained unaffected. On adding potassium to K-limited cells there was an increase in glutamate and citrulline concentrations, and a decrease in putrescine concentration, whereas concentrations of arginine and alanine remained at the very high levels observed already before addition. Resuspension of N- and K-limited cells in a complete growth medium resulted in resumption of growth, but maximum growth rate was reached only after 25-30 h. In P-limited cells resumption of growth in complete medium occurred progressively and reached the maximum rate 30 h later. P-, K- and N-limited cells resuspended into sufficient media showed different rates of ammonium and phosphate assimilation. The pattern of recovery from nutrient limitation is discussed according to the cellular role fulfilled by the nutrient which was growth rate-limiting.

The assimilation of ammonium by N-sufficient cells of unicellular algae is strictly dependent on light and CO2. However, cells previously kept under conditions of nitrogen starvation (Thacker and Syrett 1972; Syrett et al. 1986), or cells grown in chemostat culture under conditions of nitrogen limitation (Di Martino Rigano et al. 1986), can assimilate ammonium in darkness.

Chlorella sorokinianaShihira & Krauss, strain 211/8K resuspended in a illuminated mediumshowed continuous growth, and concomitantly NH4 +in the medium depleted at a constant rate. Upon sulfate removal, i) growthdeclined, ceasing within 5 h; ii) NH4 + depletionbecame almost undetectable over 2 h; and iii) photosynthetic capacity (Pc) wasreduced over 24 h by 80% in the light, but was reduced by only30% in darkness. Over 24 h of S starvation the chlorophyll content ofilluminated cells decreased by 50%, whereas that of darkened cellsdecreased by only 10%. Sulfate-deprivation over 24 h resulted in anoticeable increase in the starch content of the cell and a net increase inthe pools of glutamate, glutamine, serine and asparagine. Cysteine content, incontrast, was decreased. Sulfate addition to cells starved of S for 24 hprovoked a sudden time-dependent increase in Pc and in an immediate renewal ofgrowth in light but not in darkness. Sulphate supply also caused a suddenenhancement of respiratory oxygen consumption and a prompt degradation ofstarch. Starch was degraded at a higher rate in illuminated than in darkenedcells. After the S addition, depletion of NH4 + inthe medium occurred at slowly increasing rates; amino acids decreasednoticeably over 4 h, where in contrast, cysteine increased noticeably.

Nitrate and ammonium utilization in cell suspension of the acidophilic thermophilic non-vacuolate alga Cyanidium caldarium and the related pH variations in the external medium, were measured using ion specific electrodes. Nitrate absorption and pH variations were also monitored. The acidophilic C. caldarium allowed experimentation under acidic conditions where CO 2 exchange did not affect pH measurements. Both ammonium and nitrate assimilation occured in dark and light conditions; however, the rate of assimilation was higher in the light than in the dark. The presence of oxygen was an absolute requirement. It is suggested that the uptake of both nitrate and ammonium are mediated by active mechanisms dependent on the energy charge of the cell. The assimilation of nitrate and ammonium was accompanied by an increase and a decrease, respectively, of the external medium pH. Under steady-state conditions, for each equivalent of ammonium incorporated there were 0.8–1 equiv. of protons released into the external medium and for each equivalent of nitrate absorbed there were 1–1.2 equiv. of protons absorbed from the external medium.

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO2, assimilated NH4+ at high rates both in the light and in the dark. The assimilation of NO3−, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO2-free air, NH4+ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO2 completely restored the capacity of the alga to assimilate NH4+. NO3− assimilation, by contrast, was 33% higher in the absence of CO2 and was linear with time. It is suggested that NO3− and NH4+ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.

In the unicellular alga Cyanidium caldarium nitrate utilization is strongly inhibited by ammonium and it is resumed when ammonium has been depleted. In the presence of L‐methionine‐DL‐sulphoximine (MSX), which prevents ammonium assimilation through a specific irreversible inhibition of glutamine synthetase, nitrate reduction is no longer inhibited by ammonium, and most of the ammonium derived from nitrate reduction is excreted into the external medium. However, in the presence of MSX, nitrate reduction to ammonium proceeds at a reduced rate (45 to 70% of the control); this is particularly marked at low nitrate concentration. It is hypothesized that either MSX or accumulating ammonium bring about decrease in the rate of nitrate entry into the cell. Copyright © 1982, Wiley Blackwell. All rights reserved

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.

Assimilatory nitrate reductase and glutamate dehydrogenase of the red alga Porphyridium aerugineum have been studied. Nitrate reductase exhibits a pH optimum at approx. 7.75; it is functional with reduced benzyl viologen, FMN; FAD, NADH and is not functional with NADPH. In crude extracts of cells grown on nitrate as the sole nitrogen source, a partially inactive form of nitrate reductase occurs. This inactive enzyme can be activated by heating at 50°C. Glutamate dehydrogenase of P. aerugineum is strictly dependent on NADPH.

Summary Measurements of NH4+ uptake by Cyanidium caldarium (Tilden) Geitler, an acidophilic thermophilic non-vacuolate unicellular red alga, were made with cells grown either in batch culture with excess ammonium or in continuous culture with nitrogen limitation. Batch-grown cells absorbed NH4+ at a lower rate than chemostat-grown cells, and uptake was greatly inhibited in darkness (83 %) and by CO2 deprivation (74%). In chemostat-grown cells, in contrast, darkness inhibited uptake by only 20 to 30%. In chemostat-grown cells subjected, after the addition of ammonium, to alternate light/dark cycles over 7 h of incubation, the rate of NH4+ uptake in the successive dark periods progressively decreased and fell to only 15% of the initial rate after 5 h incubation. In the successive light periods, in contrast, there was a 15% decrease in rate from one light period to the next, and after 7 h the rate of uptake was still 50% of the initial value. At this time, transfer of the cells from light to darkness resulted in 85% inhibition of uptake. It is suggested that in C. caldarium the overall process of ammonium assimilation is controlled, at the level of ammonium entry into the cell, either through the involvement of two independently controlled uptake systems or through multiple interconvertible forms of a single system, sharing different regulatory properties.

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. © 1981 Springer-Verlag.

Summary Cyanidium caldarium is an acidophilic thermophilic red alga. At pH 1.9 with cells grown in batch culture with excess ammonium, removal of CO2 or darkness caused immediate inhibition of ammonium assimilation. By contrast, with cells grown in chemostat culture with nitrogen limitation, assimilation of ammonium was not inhibited by darkness, but it was inhibited by CO2 deprivation. In the case of these cells, inhibition was not immediate and behaved as a time-dependent phenomenon. Ammonium assimilation was restored when CO2was supplied in either light or darkness. In contrast, at pH 7.4, ammonium assimilation decreased with time both in the presence and absence of CO2, and it ceased completely after about 90 min incubation at this pH. The inhibition was not due to the high pH per se. When resuspended at pH 2, these inhibited cells were unable to assimilate ammonium during a 25 min period in the presence of CO2. Thereafter, they become able to assimilate ammonium but at a rate which was only 30% of that of the control. If they were pre-incubated at pH 2 in nitrogen-free medium for 3 h, however, they then assimilated ammonium immediately after its addition. It is suggested that a reversible inactivation at pH 1.9 and a denaturation at pH 7.4 of the system for ammonium uptake occurs, and that this uptake system is stabilized by carbon compounds against inactivation at pH 1.9 but not against denaturation at pH 7.4.

When intact cells of the acidophilic red alga Cyanidium caldarium, grown at pH 1.9 with nitrate as sole nitrogen source, are transferred to pH 7.5, they actively reduce nitrate to nitrite which accumulates in the external medium. At pH 7.5 nitrite is not further reduced to ammonia. Formation of nitrite from nitrate occurs in the light or in the dark, either in the presence or in the absence of oxygen. In cell suspensions supplied with ammonia in addition to nitrate, the formation of nitrite at pH 7.5 is strongly prevented. However ammonia is not able to prevent nitrite formation at pH 9.4. Nitrate reductase is 40% latent in cells resuspended at pH 7.5 in the presence of nitrate and up to 90% latent in cells resuspended in the presence of both nitrate and ammonia. It is proposed that the inhibition by ammonia of nitrate reduction to nitrite is based on the latent state that nitrate reductase exhibits in the presence of ammonia. In fact, at pH 9.4, when the production of nitrite is not inhibited by ammonia, nitrate reductase is only 14–25% latent even in cells resuspended in the presence of nitrate and ammonia.

A strain of Cyanidium caldarium has been studied which is able to grow in darkness using amino acids as sole energy sources. During growth ammonia was released into the external medium as a catabolic end product. With either threonine or glutamate similar rates of ammonia formation and similar kinetics of growth were observed. These observations suggest that the amounts of energy made available for cell growth from the two amino acids are equivalent. Deamination of threonine and glutamate by whole cells exhibited similar temperature-dependence profiles and similar Arrhenius energies of activation. Thus it is suggested that a partially common pathway is involved in the catabolism of these amino acids. Threonine dehydrase may play a role in this pathway. The threonine dehydrase of C. caldarium was inhibited by isoleucine and activated by valine. In the absence of isoleucine no cooperative effect of threonine was observed. Succinate or 2-ketoglutarate supported a faster growth than did amino acids. Growth tests in the presence of both a krebs cycle intermediate and an amino acid have shown that the oxidative metabolism of amino acids is in some way controlled by the more suitable energy sources, presumably through catabolite inhibition and catabolite repression. © 1977 Springer-Verlag.

Ammonium-induced inhibition of nitrate utilization was monitored in cell suspensions of the unicellular alga Cyanidium caldarium. It was found that the inhibition followed an exponential pattern with a t1/2 value of about 1.5 min in cells previously grown under conditions of excess nitrate, and of about 15 min in cells grown under conditions of severe nitrate limitation. In the latter cells only, a pretreatment with cycloheximide greatly increased the t1/2 value of inhibition. Also the resumption of nitrate utilization when ammonium was depleted followed an exponential pattern with a t1/2 value of about 4.5 min. Our results are consistent with the hypothesis that inhibition of nitrate utilization occurs at the level of nitrate reductase activity. © 1981 Springer-Verlag.

In the unicellular non-vacuolate red alga Cyanidium caldarium nitrate uptake occurs through two specific permease systems which, on the basis of kinetic constants can be defined as low affinity system and high affinity system. The high affinity system is saturated at very low nitrate concentrations (

Nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) of the unicellular alga Cyanidium caldarium can exist in two interconvertible forms; one catalytically active and one inactive. The inactive nitrate reductase can be activated by mild treatment with denaturing agents of protein. By treatment with urea or mersalyl, activation of both the NADPH and benzyl viologen activities can be realized under mild conditions, whereas by treatment with heat, the activation of benzyl viologen activity is concomitant with loss of the NADPH activity. On the other hand, both activities are activated and destroyed concomitantly by ethylene glycol. In the present of FAD, either activation of benzyl viologen activity or loss of NADPH activity upon heating occur only at higher temperatures. The existence of a controlling region in the nitrate reductase molecule is postulated. © 1980.

Chemostat cultures of the unicellular alga Cyanidium caldarium have shown that under conditions of phosphate limitation nitrate reductase is completely derepressed even in cells growing in a large excess of ammonium, but that it occurs mainly in a catalytically inactive form. It is hypothesized that phosphate limitation contributes to maintaining intracellular level of glutamine suitable to stimulate inactivation but not repression of nitrate reductase. It is not excluded that in addition to variations in the intracellular level of glutamine, there are other metabolic events of the cell by which repression and inactivation of nitrate reductase could be differently influenced. © 1984.