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.     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

Anaerobic fermentation in Cyanidium caldarium

Cyanidium caldarium cells kept anaerobically in the dark have no detectable gas exchange and form exclusively d-(-)-lactate at the expense of their starch content. The addition of acetate enhances both starch breakdown and lactate accumulation by a factor of two. During prolonged anaerobiosis Cyanidium is able to keep its energy charge at a low, but fairly constant level. The adenylate-kinase equilibrium, however, undergoes considerable changes, indicative of a regulatory mechanism which maintains a high energy charge particularly by accumlating AMP instead of ADP.     

Mechanism of aluminium tolerance in Cyanidium caldarium

Cyanidium caldarium, an acidophilic, thermophilic red alga, specifically tolerates Al. The tolerance increases at lower culture temperatures. The intracellular Al concentration is kept at low levels, especially when the cells are cultured at lower temperatures. Lower Al incorporation accounts for the Al tolerance in this alga. Fe incorporation antagonizes the Al incorporation, implying that Fe transporters incorporate Al ions. Treatment with an uncoupler, carbonylcyanide m-chlorophenylhydrazone, increases the intracellular concentration of Al. These results support the hypothesis that Al ions taken up by the algal cells are exported by an energy-dependent mechanism.     

Phycocyanobilin synthesis in the unicellular rhodophyte Cyanidium caldarium.

A method for the determination of the half-life of mitochondrial translation products in yeast in vivo is proposed. The method uses inhibitors of cytoplasmic and mitochondrial protein synthesis and is based on double-labelling pulse-chase techniques, the second label being used to estimate 'post-incorporation' during the 'chase'. For the first time the difference between post-incroporation and the widely known recycling of the label is considered. These studies show that, in the turnover of mitochondrial translation products, the problem is of post-incorporation into mitochondria (especially from the cell sap) is predominant. The results obtained with this procedure indicate that the half-life of the products of mitochondrial protein synthesis in yeast at the late-exponential phase is about 60 min. The results suggest that mitochondrial transplantation products are subject to proteolysis to acid-soluble forms.     

Studies on Cyanidium caldarium Phycobiliprotein Pigment Mutants

Phycobiliprotein biosynthesis was investigated in four strains of the unicellular rhodophyte, Cyandium caldarium, with different pigment phenotypes. All strains were incapable of synthesizing phycobiliproteins when grown in the dark. Western blotting experiments showed that dark-grown cells of the wild-type and mutant GGB synthesized the α and β subunit polypeptides of allophyocyanin and phycocyanin after exposure to light for 24 hours, whereas cells of mutant IIIC and GGBY did not. Similarly, light promoted the appearance of allophycocyanin and phycocyanin mRNAs in the wild-type and GGB but not in IIIC and GGBY. However, Southern blots of restricted genomic DNA from the wild type, IIIC, GGBY, and GGB, all hybridized with heterologous phycobiliprotein gene probes and revealed that all four strains contained identical Pst, EcoRI, and Dral restriction fragments containing allophycocyanin and phycocyanin genes. Cells of the wild type and GGB incubated in the dark with the heme precursor. δ-aminolevulinate, synthesized allophycocyanin and phycocyanin apoproteins providing strong evidence for the role of a tetrapyrrole in regulation of phycobiliprotein gene expression. However, cells of IIIC and GGBY incubated in the dark with δ-aminolevulinate did not contain detectable quantities of allophycocyanin or phycocyanin apoproteins. The possible role of a tetrapyrrole in phycobiliprotein gene expression and basis for the genetic lesion in mutants IIIC and GGBY is discussed.     

Revision of Cyanidium caldarium. Three species of acidophilic algae

Abstract

The authors carry out a systematic revision of three unicellular eucaryotic algae, often living in mixed population in thermal acidic environment. Such algae were often confused under the binomium Cyanidium caldarium.

The authors state that the following specific binomia are to be attributed to the three algae: Galdieria sulphuraria (Galdieri) Merola comb. nova; Cyanidium caldarium Geitler non (Tilden) Geitler emend.; Cyanidioschyzon merolae De Luca, Taddei & Varano.

The family Galdieriaceae is instituted for the first of these algae, whereas the other two algae are included in the family Cyanidiaceae Geitler emend.

The class Cyanidiophyceae Merola, a new class of the Rhodophyta, is instituted for these two families.     

Evidence for two transport systems for nitrate in the acidophilic thermophilic alga Cyanidium caldarium

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 (<1 M), whereas the low affinity system is saturated only at high nitrate concentrations (K _m=0.45±0.10 mM). The low affinity system is present in cells growing under conditions of nitrogen limitation as well as in cells growing in excess nitrate. In contrast, the high affinity system is present only in cells growing under conditions of nitrogen limitation. The high affinity system works only at acid pH and is inactive at neutral pH. The low affinity system is active both at acid and at neutral pH.     

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.     

Electron donors and inhibitors of nitrate reductase from Cyanidium caldarium.

Studies on nitrate reductase (NAD(P)H:nitrate oxidoreductases EC 1.6.6.2) of Cyanidium caldarium revealed that the enzyme is inhibited by excess of electron donor, NADPH, reduced benzylviologen and FMN. Also dithionite, used to reduce benzylviologen and FMN, inactivates nitrate reductase: however, FMN at an optimal concentration and nitrate, added before the dithionite, protect the enzyme against this inactivation. Cyanide, cyanate and carbamyl phosphate inhibit the enzyme competitively with respect to nitrate, and Ki values are reported. Organic mercurials, 0.1 mM, act preferentially on NADPH activity, whereas Ag+ and Hg-2+ at the same concentration inactivate 80--90% of the benzylviologen and FMN activities. ADP is very poor inhibitor. Urea 4 M in 2 h destroys 90% of the NADPH activity and only 30% of the benzylviologen and FMN activities. The apparent Km values for NADPH, benzylviologen, FMN and nitrate have been determined.     

Biosynthesis of phycocyanobilin from exogenous labeled biliverdin in Cyanidium caldarium

Phycocyanin is a major light-harvesting pigment in bluegreen, red, and cryptomonad algae. This pigment is composed of phycocyanobilin chromophores covalently attached to protein. Phycocyanobilin is an open-chain tetrapyrrole structurally close to biliverdin. Biliverdin is formed in animals by oxidative ring-opening of protoheme. Recent evidence indicates that protoheme is a precursor of phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium. To find out if biliverdin is an intermediate in the conversion of protoheme to phycocyanobilin, [14C]biliverdin was administered along with N-methylmesoporphyrin IX (which blocks endogenous protoheme formation) to growing cells of C. caldarium. To avoid phototoxic effects due to the porphyrin, a mutant strain was used that forms large amounts of both chlorophyll and phycocyanin in the dark. After 12 or 24 h in the dark, cells were harvested and exhaustively extracted to remove free pigments. Next, protoheme was extracted. Phycocyanobilin was then cleaved from the apoprotein by methanolysis. Protoheme and phycocyanobilin were purified by solvent partition, DEAE-Sepharose chromatography, and preparative reverse-phase high-pressure liquid chromatography. Absorption was monitored continuously and fractions were collected for radioactivity determination. Negligible amounts of label appeared in the protoheme-containing fractions. A major portion of label in the eluates of the phycocyanobilin-containing samples coincided with the absorption peak at 22 min due to phycocyanobilin. In a control experiment, [14C]biliverdin was added to the cells after incubation and just before the phycocyanobilin-apoprotein cleavage step. The major peak of label then eluted with the absorption peak at 12 min due to biliverdin, indicating that during the isolation biliverdin is not converted to compounds coeluting with phycocyanobilin. It thus appears that exogenous biliverdin can serve as a precursor to phycocyanobilin in C. caldarium, and that the route of incorporation is direct rather than by degradation and reincorporation of 14C through protoheme.     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.