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.     

Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

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.     

Incorporation of haem into phycocyanobilin in levulinic acid treated Cyanidium caldarium

Cells of the red alga Cyanidium caldarium were preincubated with 5 mmol 1^–1 levulinic acid (LA) and subsequently incubated with ¹⁴C-labelled haem (5.67 Bq nmol^–1). Phycocyanin was isolated. The specific radio-activity of its chromophore phycocyanobilin (PCB) was determined after cleavage and purification by thin layer chromatography. The percentage of PCB formed from labelled haem within 0.5 h was considerably higher in LA treated cells than in non-treated controls. This difference disappeared after prolonged incubation (16.5 h) with haem. The results are interpreted as possible incorporation of haem into preexisting apoprotein.Abbreviations LA levulinic acid - PCB phycocyanobilin     

Respiration-Dependent H+ Efflux from Intact Cells of Cyanidium caldarium

An active H⁺ efflux depending on respiration was found in anacidophilic unicellular alga, Cyanidium caldarium. Alkalizationof the medium due to passive H⁺ transport into the cells wasobserved when the respiratory activity was inhibited by addingrespiratory poisons, such as rotenone or antimycin A, or byintroducing pure nitrogen into the cell suspension. The extentof the H⁺ influx increased as the pH of the medium was loweredto 2.9, indicating that H⁺ leaks into the cells according tothe pH gradient across the plasma membrane. The medium pH whichhad increased under anaerobic condition returned to the originallevel with aeration of the cell suspension. This suggests thatan active H⁺ transport, related to respiration, pumps out theexcess H⁺ accumulated in the cells during anaerobic preincubation.The pH changes in the cell suspension were related to the intracellularATP level. From these results it was concluded that active H⁺efflux dependent upon oxidative phosphorylation functions inthe dark to maintain a constant intracellular pH against passiveH⁺ leakage through the plasma membrane. The light-induced H⁺ efflux and the respiration-dependent H⁺efflux were also compared in relation to the physiological roleof the active H⁺ efflux, especially with respect to the intracellularpH regulation in this alga. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

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.     

Light-induced H+ Efflux from Intact Cells of Cyanidium caldarium

Light-induced pH changes in suspensions of an acidophilic unicellularalga, Cyanidium caldarium Geitler, were studied as a functionof the pH of the medium. In the neutral pH region, alkalizationof the medium due to photosynthetic CO₂ uptake was observed.In the acidic pH region, illumination caused a significant decreasein the pH of the medium, indicating the efflux of H⁺ from thecells. Both the rate and extent of the pH decrease increasedas the pH of the medium was lowered to 3.0. The H⁺ efflux wasnot affected by 3-(3',4'-dichlorophenyl)-l,l-dimethylurea, butwas inhibited by phenylmercuric acetate. The fastest H⁺ effluxoccurred at 45°C, whereas its extent was almost constantfrom 25 to 50°C. The activity decreased at temperaturesabove 50°C and was inactivated completely at 60°C. Itsaction spectrum corresponded the spectrum for chlorophyll aabsorption. Results indicate that the light-induced H⁺ effluxis driven by photosystem I and is important in the maintenanceof the intracellular pH at the functional neutral region againsta steep pH gradient across the cell membrane. (Received May 6, 1981; Accepted August 8, 1981)     

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.     

Chloroplast Nucleoids during Greening of Cyanidium caldarium M-8 Type

Cyanidium caldarium M-8 type grown in the dark was illuminatedfor 3 days, and changes of its cell and cell organelle structuresand of photosynthetic activity were observed quantitatively.Dark grown (DG) cells showed no photosynthetic activity andno phycocyanin. During 3 day illumination they fully recoveredtheir photosynthetic activities as measured by Hill reaction,and also synthesized chlorophyll a and phycocyanins. Sizes ofcell, cell nucleus, chloroplast and its nucleoid observed byfluorescence microscopy after staining with DAPI increased simultaneouslyupon illumination. The chloroplast and its ring shaped nucleoidsizes increased especially rapidly, concomitant with the recoveryof Hill activity. In fully recovered cells after 3 days, a goodcorrelation was found among the sizes of cells, chloroplastsand chloroplast nucleoids. ( Revision received June 28, 1986. Accepted December 23, 1986)     

Physical-chemical properties of C-phycocyanin isolated from an acido-thermophilic eukaryote, Cyanidium caldarium.

C-Phycocyanin from an acido-thermophilic eukaryotic alga, Cyanidium caldarium, was characterized with respect to subunit structure, absorption spectrum and fluorescence properties and was found to be similar to C-phycocyanins from mesophilic sources. The pH-dependence of fluorescence polarization and the changes in sedimentation velocity as a function of pH, concentration and temperature indicate the presence of extremely large amounts of unusually stable 19S aggregates. It was not possible to disaggregate this phycocyanin completely to monomer under normal conditions. The amino acid composition is similar to that of phycocyanins from other thermophilic and halophilic sources. The isoelectric point of this C-phycocyanin was 5.11, an unusually high value. The properties of this C-phycocyanin suggest an increase in protein stability as its mode of adaptation to the environmental stress of high temperature.     

Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat

C-Phycocyanin, a biliprotein, was purified from the red alga, Cyanidium caldarium. This alga grows at temperatures up to 57 degrees C, a very high temperature for a eukaryote, and at pH values down to 0.05. Using the chromophores on C-phycocyanin as naturally occurring reporter groups, the effects of temperature on the stability of the protein were studied by circular dichroism and absorption spectroscopy. The protein was unchanged from 10 to 50 degrees C, which indicates that higher temperatures are not required to cause the protein to be photosynthetically active. At 60 and 65 degrees C, which are above the temperatures at which the alga can survive, the protein undergoes irreversible denaturation. Gel-filtration column chromatography demonstrated that the irreversibility is caused by the dissociation of the trimeric protein to its constitutive polypeptides. Upon cooling, the alpha and beta polypeptides did not reassemble to the trimer. Unlike phycocyanins 645 and 612, the C-phycocyanin does not show a reversible conformational change at moderately high temperatures. At constant temperature, the C-phycocyanin was more stable than a mesophilic counterpart. It is designated a temperature-resistant protein.     

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.