Amodio Fuggi

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.

Glutamine synthetase activity, ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.

Nitrate and ammonium assimilation in algal cell-suspensions and related pH variations in the external medium, monitored by electrodes

Abstract 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.

Studies on utilization of 2-ketoglutarate, glutamate and other amino acids by the unicellular alga Cyanidium caldarium.

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.

Studies in vivo on the control by ammonia of nitrate reduction to nitrite in the unicellular alga Cyanidium caldarium

Abstract 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.

Heterotrophic growth patterns in the unicellular alga Cyanidium caldarium

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.

Mechanism of proton-linked nitrate uptake in Cyanidium caldarium, an acidophilic non-vacuolated alga

Abstract The unicellular non-vacuolated alga Cyanidium caldarium, grown under conditions of nitrogen limitation, possesses two permease systems for nitrate uptake, one of which, the so-called ‘high-affinity nitrate uptake system’, enables the alga to take up nitrate through a mechanism involving cotransport of protons. Measurements of nitrate and proton stoichiometry, and determination of the kinetic parameters of uptake in cells resuspended in medium adjusted at different pH values, are consistent with a mechanism of uptake in which two protons for each nitrate ion are transported across the plasmalemma. Furthermore, kinetic data suggest that the carrier first binds nitrate and, subsequently, protons. Permutations of this binding sequence do not agree with the experimental results.

Regulation of Nitrate Reductase in Chlorella. Nitrate Requirement for the Appearance of Nitrate Reductase Activity

Summary Cells of Chlorella vulgaris (Cambridge culture collection, strain 211/12) grown in chemostat under conditions of nitrate limitation, exhibit very high levels of nitrate reductase activity. Similar levels of activity were found in cells grown in chemostat with a limited nitrogen source constituted by 75% ammonium plus 25% nitrate. By contrast, cells grown under conditions of ammonium limitation exhibit very low levels of nitrate reductase activity. Cells grown under conditions of excess nitrate have only 15-20% of the activity found in cells grown under conditions of nitrate limitation and under conditions of excess ammonium, even in the presence of nitrate, they have no appreciable nitrate reductase activity. It is emphasized that in the C. uulgaris strain studied here the appearance of nitrate reductase activity requires not only the absence of an excess amount of ammonium, but also the presence of nitrate. Cells grown under conditions of either ammonium excess or limitation attain full capacity to assimilate nitrate after 150-min exposure to nitrate. During this 150-min period the alga assimilated nitrate at increasing rates.

Pattern of inhibition of nitrate utilization by ammonium in the acidophilic thermophilic unicellular alga Cyanidium caldarium

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.

Short-term regulation of nitrate uptake by a ‘pump and leak’ mechanism in the acidophilic nonvacuolated alga, Cyanidium caldarium

In the nonvacuolated acidophilic thermophilic red alga, Cyanidium caldarium , nitrate uptake and reduction can be separated by measuring disappearance of nitrate from the suspension medium using, in in vivo experiments, cyanate, a competitive inhibitor of algal nitrate reductase. Cyanate selectively inhibited nitrate reduction at concentrations that did not significantly affect nitrate uptake, photosynthesis or respiration. Its use proved that short-term control of intracellular nitrate through the increase of a carrier-mediated nitrate efflux took place when nitrate reduction was inhibited. The occurrence of the high- and low-affinity nitrate uptake systems in cells grown in nitrogen-limited conditions, as previously reported, suggests a ‘pump and leak’ mechanism operating at the plasmalemma level to regulate nitrate uptake and intracellular nitrate: the high-affinity nitrate transport system mediated by proton cotransport (irreversible) operates the influx, while the low-affinity transport (reversible) operates influx or efflux according to cell requirements. Kinetic analysis of cyanate inhibition in cells taken from low-nitrate medium supports this hypothesis and reveals that, in Cyanidium , intracellular nitrate is probably compartmented in the cytosol.