A Comparative Ultrastructural Study of Cyanidium caldarium and the Unicellular Red Alga Rhodosorus marinus

The presence of nucleus, chloroplasts, mitochondria, microbodiesand a vacuole are confirmed in Cyanidium caldarium strain CCAP1355/1. Chloroplasts usually contain parallel rows of unstackedthylakoids surrounded by a peripheral thylakoid, although theconcentric arrangement has also been observed. The chloroplastenvelope consists of two closely appressed membranes which canonly be resolved after gluteraldehyde—osmium fixation.The chloroplast of Rhodosorus marinus also contains parallel,unstacked thylakoids surrounded by a peripheral thylakoid but,in addition, a prominent, stalked pyrenoid projects into thecytoplasm and is bounded by both the peripheral thylakoid andthe chloroplast envelope. This structure is covered by a capof starch grains and contains membranous vesicles in the matrix.Phycobilisomes are absent from the chloroplasts of both algae.The recognition of C. caldarium as a rhodophyte is supportedby these observations. Cyanidium caldarium, Rhodosorus marinus, chloroplast ultrastructure     

Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium

Cell-free extracts of the unicellular red alga Cyanidium caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low molecular weight components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation, and mesobiliverdin cannot serve as a substrate.

Incubation of cell extracts with biliverdin yields two products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same two products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The two products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z- and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliverdin transformation is therefore the Z-ethylidine isomer. The time course for formation of the two products during incubation suggests that the early-eluting product is the precursor of the late-eluting one. These results suggest that Z-ethylidine phycocyanobilin is the precursor of the E-ethylidine isomer, and that the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore.

     

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.     

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)     

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.     

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.     

External Iron Regulates Polyphosphate Content in the Acidophilic, Thermophilic Alga Cyanidium caldarium

Transmission electron microscopy revealed the presence of electron-dense bodies (EDB) in the cytosol of the acidophilic, thermophilic red alga Cyanidium caldarium. These bodies contain almost exclusively Fe, P, and O and can play a role in Fe storage. ³¹P-nuclear magnetic resonance analysis identified a sharp signal at −23.3 ppm, which was attributed to the phosphate groups of the inner portions of polyphosphate chains. From this evidence, as well as that of a previous ESR study (Nagasaka et al., BioMetals 16:465–470, 2003), it can be concluded that polyphosphates are the major anionic constituents of the EDB. Omission of Fe from the culture medium resulted in substantially decreased polyphosphate levels, demonstrating the control of cellular polyphosphate content by the Fe status of the culture medium.     

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     

Biosynthesis of phycobilins. Ferredoxin-supported nadph-independent heme oxygenase and phycobilin-forming activities from Cyanidium caldarium.

The unicellular red alga, Cyanidium caldarium, synthesizes phycocyanobilin from protoheme via biliverdin IX alpha. In vitro transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins were previously shown to require NADPH, ferredoxin, and ferredoxin-NADP+ reductase, as well as specific heme oxygenase and phycobilin formation enzymes. The role of NADPH in these reactions was investigated in this study. The C. caldarium enzymatic activities that catalyze biliverdin IX alpha formation from protoheme, and phycobilin formation from biliverdin IX alpha, were partially purified by differential (NH4)2SO4 precipitation. The enzyme fractions, when supplemented with a light-driven ferredoxin-reducing photosystem I fraction derived from spinach leaves, catalyzed light-dependent transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins, with or without the addition of NADPH and ferredoxin-NADP+ reductase. In the dark, neither reaction occurred unless NADPH and ferredoxin-NADP+ reductase were supplied. These results indicate that the only role of NADPH in both reactions of phycobilin biosynthesis, in vitro, is to reduce ferredoxin via ferredoxin-NADP+ reductase and that reduced ferredoxin can directly supply the electrons needed to drive both steps in the transformation of protoheme to phycocyanobilin.     

Photochemical Performance of the Acidophilic Red Alga Cyanidium sp. in a pH Gradient

The acidophilic red alga Cyanidium sp. is one of the dominant mat-forming species in the highly acidic waters of Río Tinto, Spain. The culture of Cyanidium sp., isolated from a microbial mat sample collected at Río Tinto, was exposed to 9 different pH conditions in a gradient from 0.5 to 5 for 24?h and its physiological status evaluated by variable chlorophyll a fluorescence kinetics measurements. Maximum quantum yield was determined after 30?min, 1?h, 2?h, 4?h, 6?h and 24?h of exposure after 15?min dark adaptation. The effect of pH on photochemical activity of Cyanidium sp. was observable as early as 30?min after exposure and the pattern remained stable or with only minor modifications for 24?h. The optimum pH ranged from 1.5 to 2.5. A steep decrease of the photochemical activity was observed at pH below 1 even after 30?min of exposure. Although the alga had tolerated the exposure to pH?=?1 for at least 6?h, longer (24?h) exposure resulted in reduction of the photochemical activity. At pH above 2.5, the decline was more moderate and its negative effect on photochemistry was less severe. According to the fluorescence measurements, the red alga Cyanidium sp. is well-adapted to prevailing pH at its original locality at Río Tinto, i.e. pH of 1 to 3. The short-term survival in pH?相似文献   

Biosynthesis of phycobiliproteins. Incorporation of biliverdin into phycocyanin of the red alga Cyanidium caldarium

14C-labelled biliverdin IX alpha was administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Between 9 and 12% of the phycobiliprotein chromophore produced in such cultures was derived from exogenous biliverdin. These results demonstrate that biliverdin is an intermediate in the biosynthesis of phycobiliproteins.     

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.     

Photo- and Metabolite Regulation of the Synthesis of Ribulose Bisphosphate Carboxylase/Oxygenase and the Phycobiliproteins in the Alga Cyanidium caldarium

In the eukaryotic and unicellular alga Cyanidium caldarium the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and the light gathering proteins phycocyanin (PC) and allophycocyanin (APC) is under the control of light and glucose, which is a metabolizable carbon source for this organism. Light promotes the synthesis of these proteins while glucose has a strong inhibitory effect on this process. All subunits of the proteins mentioned above are in vitro translation products of poly (A)⁻-RNA (Steinmüller, Kaling, Zetsche 1983 Planta 159: 308-313). Both factors—light and glucose—exert their effects mainly by modulation of the level of translatable messenger RNA for these proteins. Under autotrophic growth conditions the level of translatable RuBPCase-, PC-, and APC-messenger RNA is high, whereas in the presence of glucose the level of these mRNAs is low or not detectable at all.     

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.     

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)     

Characterization of the Nuclear- and Plastid-Encoded secA-Homologous Genes in the Unicellular Red Alga Cyanidioschyzon merolae

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.     

Photosynthetic oxygen production and the size of the photosynthetic unit in a cell-free preparation from Cyanidium caldarium

A cell-free preparation has been isolated from a mutant of Cyanidium caldarium, grown under conditions such that there is 15 times less chlorophyll per photosynthetic unit than in normal green algae. The preparation is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and shows the well-characterized oscillation of O₂ yield, from saturating flashes, following a period of dark adaptation. Greening experiments with dark-grown, wild-type Cyanidium show that the synthesis of photosynthetic units precedes that of bulk chlorophyll and that the O₂-producing system is assembled before the total system coupled to CO₂. No large-scale cooperation of chlorophyll molecules is required for O₂ production.