Intracellular Localization of Enzymes of Fatty Acid-beta-Oxidation in the Alga Cyanidium caldarium

The intracellular distribution of enzymes, participating in the β-oxidation of fatty acids in the eucaryotic alga Cyanidium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and thiolase were present in the mitochondrial fraction (density: 1.19 gram per cubic centimeter). Activity of an acyl-CoA synthetase was found in the mitochondrial fraction as well as in a band where mitochondrial membrane apparently had accumulated (density: 1.17 gram per cubic centimeter). None of these enzymes were present in the peroxisomes (density: 1.23 gram per cubic centimeter). Results from cell fractionation as well as properties of β-oxidation enzymes indicate a mitochondrial location of fatty acid degradation also in the algae Galdieria sulphuraria and Cyanidioschyzon merolae.     

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.     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

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.     

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.     

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.     

Effect of Intracellular ATP Levels on the Light-Induced H+ Efflux from Intact Cells of Cyanidium caldarium

The light-induced H⁺ efflux observed at acidic pH in Cyanidiumcells was shown to be an active H⁺ transport depending on theintracellular ATP produced by cyclic photo-phosphorylation.Triton X-100 was found to act as an effective uncoupler in intactCyanidium cells without collapsing the pH gradient across theplasma membrane. Triton X-100 at 0.015% significantly reducedthe intracellular ATP levels, stimulated the p-BQ, Hill reactionand completely inhibited the light-induced H⁺ efflux. Inhibitionof the H⁺ efflux by Triton X-100 correlated well with the depressionof the apparent rale of light-induced ATP synthesis as wellas the decrease in the intracellular ATP level in light. The light-induced H⁺ efflux was completely inhibited by diethylstilbestrol,a specific inhibitor of plasma membrane ATPase, without anychanges in the intracellular ATP level, thereby suggesting theparticipation of the plasma membrane ATPase in the light-inducedH⁺ efflux. 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)     

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.     

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.     

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)     

Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium

The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine-glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed.     

Recovery of metals from acid waste water by Cyanidium caldarium

Summary Cyanidium caldarium grows in acid water obtained from a bacterial leaching procedure and the alga is capable of precipitating metals from the solution. This study demonstrates that changes in culture and oxidation-reduction conditions may result in a different bioaccumulation of metals.     

Thermostability of the Photosynthetic System of the Thermoacidophilic Alga Cyanidium caldarium in Continuous Culture

Ford, T. W. 1986. Thermostability of the photosynthetic systemof the thermoaadophilic alga Cyanidium caldarium in continuousculture.—J. exp. Bot. 37: 1698–1707. Cyanidium caldarium, when exposed to gradual increases in temperaturein continuous culture, exhibits a growth temperature maximumof 55 °C. This correlates with the thermostability of themembrane-located photosynthetic electron transport system butnot with in vivo ribulose-1,5-bisphosphate (RUBP) carboxylaseactivity, which retained full activity after 1 h at 60 °C.Pigment content and phycocyanin: chlorophyll a ratios were relativelyconstant at growth temperatures up to 50 °C, but both declinedin cells cultured at 55 °C. Some modification of the photosyntheticsystem of the alga, in response to growth temperature, was detectedwith both oxygen evolution and RUBP carboxylase activity showingimproved thermostability in cells grown at 50 °C or 55 °Ccompared with those cultured at lower temperatures. However,this enhanced thermostability was at the expense of total pigmentcontent and overall photosynthetic capacity which were considerablyreduced in high temperature cells, as was the temperature spectrumfor efficient RUBP carboxylase operation. The contributionsof membrane and macromolecular components of the cell to theimposition of optimum and maximum growth temperatures are discussed. Key words: Cyanidium, photosynthesis, RUBP carboxylase