FLORIDOSIDES IN CYANIDIUM CALDARIUM,CYANIDIOSCHYZON MEROLAE AND GALDIERIA SULPHURARIA(RHODOPHYTA,CYANIDIOPHYCEAE)1

Cyanidium caldarium Geitler, Cyanidioschyzon merolae De Luca, Taddei & Varano and Galdieria sulphuraria (Galdieri) Merola are the three thermoacidophilic algae characterized by a chloroplast which is bounded by a single membrane. The presence of this atypical chloroplast made the inclusion of these algae in the Rhodophyta difficult. The discovery in the three algae of floridoside and isofloridoside, typical storage products of red algae, in compatible with their inclusion in the Rhodophyta     

Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D63675 and D63676.     

In-vitro synthesis of phycobiliproteids and ribulose-1,5-bisphosphate carboxylase by non-poly-adenylated-RNA of Cyanidium caldarium and Porphyridium aerugineum

Polyadenylated and nonpolyadenylated mRNa from the red algae Cyanidium caldarium Geitler and Porphyridium aerugineum Geitler were translated in a cell-free system. The α-and β-subunits of phycocyanin and allophycocyanin and also both subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) were identified with the help of specific antibodies as translation products of non-polyadenylated mRNA. Both subunits of RuBPCase were synthesized with molecular weights in the range of the mature forms. This is in contrast to the findings with green algae and higher plants where the small subunits of RuBPCase are always made by polyadenylated mRNA in the form of a larger precursor molecule. We discuss our findings with regard to the systematic position of the algae used and the evolution of plastids.     

Evaluation of toxic effects of heavy metals on unicellular algae. V-Influence of extracellular products on toxicity and on type of inhibition

Abstract

Results suggest that the toxicity of mercury and copper to two unicellular algae (Cyanidium caldarium and Chlorella saccharophila) can be decreased either through their subtraction from the culture medium by living or dead cells, or by the extracellular products.

The subtraction of the heavy metals tested manifests itself in each case by a shortening of the lag phase. Further, at least in the case of the combination Chlorella saccharophila-copper, a transition from type III inhibition (increased lag phase) to type II (decreased growth rate) as a results of an increase in the concentration of copper in the presence of extracellular products was observed.

This phenomenon was explained by the different toxic action exerted by copper in its ionic form (type III) as contrasted with copper combined in metalorganic compounds (type II).     

Molecular variation in Galdieria sulphuraria (Galdieri) Merola and its bearing on taxonomy

Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria are three unicellular algae characteristic, of acid thermal environments. Recently, on the basis of morphological characters, three new species of Galdieria (G. partita, G. daedala, G. maxima ) isolated from acid-thermal springs in Russia have been instituted. A selected region of rbcL and the sequence of the intergenic spacer between the rbcL and rbcS have been amplified and sequenced from different Galdieria species and strains, in order to define molecular relationship among these interesting algae. The obtained cladogram shows that Cyanidium caldarium and Cyanidioschyzon merolae form a sister group which, in turn, is in a sister group relationship with Galdieria. This last genus is divided in two clades, one of which includes G. sulphuraria accessions from Naples (Italy), California, and Yellowstone and the other one includes G. sulphuraria accessions from Java (Indonesia) and from the Russian species. These results support the status of the genus Galdieria and suggest that G. daedala, G. maxima and G. partita are three very similar strains of G. sulphuraria; the rbcL variation within Galdieria accessions has a pattern which is broadly connected to the geographial distribution. The data obtained from the intergenic rbcL-rbcS spacer partly confirm those from the rbcL analysis.     

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.     

Lipid composition of cyanidium

The major lipids in Cyanidium caldarium Geitler are monogalactosyl diglyceride, digalactosyl diglyceride, plant sulfolipid, lecithin, phosphatidyl glycerol, phosphatidyl inositol, and phosphatidyl ethanolamine. Fatty acid composition varies appreciably among the lipids, but the major ones are palmitic acid, oleic acid, linoleic acid, and moderate amounts of stearic acid. Trace amounts of other acids in the C₁₄ to C₂₀ range were also present. Moderate amounts of linolenic acid were found in two strains, but not in a third. The proportion of saturated acid is relatively high in all lipids ranging from about a third in monogalactosyl diglyceride to three-fourths in sulfolipid. This may be a result of the high growth temperature. Lipases forming lysosulfolipid, and lysophosphatidyl glycerol are active in ruptured cells; galactolipid is degraded with loss of both acyl residues. Thus the lipid and fatty acid composition of Cyanidium more closely resembles that of green algae than that of the blue-green algae, although there are differences of possible phylogenetic interest.     

Structural features of the plastid ribosomal RNA operons of two red algae: Antithamnion sp. and Cyanidium caldarium

The nucleotide sequences of the plastid 16S rDNA of the multicellular red alga Antithamnion sp. and the 16S rDNA/23S rDNA intergenic spacers of the plastid DNAs of the unicellular red alga Cyanidium caldarium and of Antithamnion sp. were determined. Sequence comparisons support the idea of a polyphyletic origin of the red algal and the higher-plant chloroplasts. Both spacer regions include the unsplit tRNA^Ile (GAU) and tRNA^Ala (UGC) genes and so the plastids of both algae form a homogeneous group with those of chromophytic algae and Cyanophora paradoxa characterized by small-sized rDNA spacers in contrast to green algae and higher plants. Nevertheless, remarkable sequence differences within the rRNA and the tRNA genes give the plastids of Cyanidium caldarium a rather isolated position.     

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     

Cyanidium caldarium as a bridge alga between Cyanophyceae and Rhodophyceae: Evidence from immunodiffusion studies

The primer-independent phosphorylase isozyme, a₂, of Cyanidiumcaldarium was used for immunization of rabbits. The immune serumwas tested against pure a₂ isozymes from blue-green, red, andgreen algae. Double immunodiffusion in agar indicated that therewas structural similarity in the isozyme from Cyanidium caldarium,the blue-green algae, Oscillaloria princeps, Pleclonema nostocorumand the red alga, Rhodymenia pertusa. Complete fusion of theprecipitin lines was obtained with these algae. However, onlypartial fusion was observed with the a₂ isozymes from Chlorophyceaesuch as Chlorella pyrenoidosa and Spirogyra setiformis. Spurformation on the precipitin lines occurred when the isozymesfrom these algae were tested against the immuneserum. The results were interpreted as indicative of the possible transitionstatus of Cyanidium caldarium between prokaryotic blue-greenalgae and eukaryotic red algae. It would appear that the Chlorophyceaeevolved along different lines from Cyanophyceae than did theRhodophyceae. (Received November 25, 1975; )     

Low molecular weight carbohydrates in Cyanidium caldarium and some related algae

Floridoside (2-O-glycerol-α-d-galactopyranoside) and a small amount of iso-floridoside (1-O-glycerol-α-d- galactopyranoside) were found in Cyanidium caldarium. Floridoside was also found in the red algae Porphyridium cruentum and Porphyra yezoensis, although in the latter iso-floridoside was the main component. Sucrose and glucose were found in the green algae Chlorella pyrenoidosa and Scenedesmus obliquus, and also in a blue-green alga, Anacystis nidulans. Another blue-green alga, Phormidium foveolarum, contains mostly trehalose. From these results and from morphological considerations, it is suggested that Cyanidium caldarium belongs to the primitive Rhodophyta.     

WATER STATUS AND THE DISTRIBUTION OF CYANIDIUM CALDARIUM IN SOIL1

The size and photosynthetic ability of Cyanidium caldarium populations in soil were related to the water status of the soil, welter soil, areas having larger populations and more extensive photosynthesis. The application of the direct methods used to the study of other soil algae, mosses, and lichens is discussed.     

Polyglucan Branching Isoenzymes of Algae

The cyanophyte, Oscillatoria princeps, and the enigmatic hot-spring alga, Cyanidium caldarium, contain two branching isoenzymes which can act on amylose and amylopectin, converting these polyglucans into highly branched phytoglycogens. The red alga, Rhodymenia pertusa contains three branching isoenzymes, only one of which is capable of the dual activity of the other algal branching isoenzymes. The other two red algal branching isoenzymes are Q enzymes and can only act on amylose, forming moderately branched amylopectìns. However, when the isoenzymes of all three algae are separated on different concentrations of polyacrylamide gel via electrophoresis, the mobilities of the isoenzymes show that the Q enzymes and the branching enzymes are related and true isoenzymic molecules. They differ only in electrical charges, probably caused by the substitution of amino acid residues in their active peptides. It is possible that if these charge isomers are due merely to amino acid substitutions, the enzymes may have been originally derived from a common catalytic molecule. The study indicates the identity of the branching isoenzymes of Oscillatoria and Cyanidium, and lends further supporting evidence to the cyanophyte origin of the enigmatic alga.     

THE ALGAE OF WAIMANGU CAULDRON (NEW ZEALAND): DISTRIBUTION IN RELATION TO pH1

The pH of the main body of thermal water and of the outlet of Waimangu Cauldron (Frying Pan Lake) is 3.8, whereas hot springs flowing into this lake are alkaline, pH 8.2–8.7. Where these waters meet there are pH gradients, and it is possible to find a series of habitats differing in pH but having approximately the same temperature (54–56 C). Where the pH is above 4.8 the blue-green alga Mastigocladus laminosus is present, and where the pH is below this value the eucaryotic alga Cyanidium caldarium is the sole photosynthetic microorganism at temperatures less than 55 C. Rocks resting on the bottom of Waimangu Cauldron surrounded by water of pH 3.8 have Mastigocladus laminosus growing on them, but measurements of pH directly in these algal mats showed that the microenvironment of the alga was actually above pH 4.8. A simple experiment showed that it is probably the rock, per se, that initiates a tiny island of alkalinity in the acid lake, permitting M. laminosus to become established. The outlet of Waimangu Cauldron is a hot acid stream which becomes progressively alkaline as it descends through Waimangu Valley, due to inflow of alkaline water from springs and a cold stream. Blue-green algae were found in this stream when the pH was greater than 5.0 and C. caldarium when the pH was less than 4.0. In one region where the pH was between 4.0 and 5.0, both blue-green algae and C. caldarium were seen, although in moribund state. These data, provide further information on the lowest pH values at which blue-green algae are able to grow.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Storage glucan biosynthesis in Cyanidium, Chlorella and Prototheca: evidence for endosymbiosis?

The storage glucans of Chlorella pyrenoidosa and Prototheca zopfii are identical and consist of a linear polyglucan akin to amylose and a branched amylopectin component. The branched glucans of these algae differ markedly from that formed by the hot-springs alga, Cyanidium caldarium. The more highly branched Cyanidium glucan appears to be formed by branching glucosyltransferases which are different from those of the other two algae. The relevance of the data to the possibility of Cyanidium being a Prototheca-like Chlorella that has acquired symbiotic Cyanobacteria as chloroplasts is discussed.     

Differences of the primer-independent phosphorylase isozyme in thermophilic and mesophilic algae

Kinetic studies of the primer-independent, aa phosphorylaseisozyme of two thermophilic algae, Cyanidium caldarium and Oscillatoriaprinceps, and two mesophilic algae, Spirogyra setiformis andChlorella pyrenoidosa, indicate that the isozyme of thermophilicspecies is more resistant to denaturation by heat, and has lessaffinity for the glucose-1-phosphate substrate than the sameisozyme of mesophilic algae. This points out the possibilitythat this enzyme's structure is more rigid in thermophiles andmay lack the flexible conformation found in the mesophilic isozyme.The evolutionary significance of thermophily as a primitivecharacter rather than an adaptive trait is discussed in thelight of studies with a low temperature variant strain of thethermophilic alga, Oscillatoria princeps, LTV. (Received November 15, 1972; )     

Intracellular Regime for the Assembly of the Chlorophyll-Protein Complex CPI in Cyanidium caldarium

Mass cultures of Cyanidium caldarium exhibit a developmentalstage of temporary bleaching during cultivation in permanentlight (Zagon and Brandt, 1985). This loss of chlorophyll a isnot accompanied by a loss of the CPI-apoprotein. This essentialthylakoid protein is also inserted into the thylakoid membranewithout the presence of chlorophyll a. Subsequent assembly withthe pigment, however, increases the intrinsic binding of theCPI-apoprotein. The temporary bleaching of C. caldarium canbe imitated essentially by addition of levulinic acid or cycloheximide,but not by chloramphenicol or streptomycin. The cooperationof the nucleocytoplasm and of the photosyn-thetically activecompartments with respect to the assembly of CPI is discussed. Key words: Assembly, chlorophyll-protein complex, Cyanidium caldarium, regulation     

Occurrence of 2-hydroxy acids in microalgae

2-Hydroxy acids were believed to be absent in algae until this study, in which the analysis of microalgae belonging to Chlorophyta (Chlamydomonas reinhardtii and Chlorella pyrenoidosa), Rhodophyta (Cyanidium caldarium M-8 and Cyanidium caldarium RK-1) and Cyanophyta (Anbaena variabilis, Anacystis nidulans, Oscillatoria species and Phormidium foveolarum) is reported. 2-Hydroxy adds with carbon chain lengths of C₁₆-C₂₆, were found in all the algal samples studied, ranging in concentrations from 4.0 to 320μg/g dry alga. The dominant constituents are 2-hydroxyhexadecanoic, 2-hydroxynonadecanoic, 2-hydroxyhexacosanoic and a branched 2-hydroxy-C₁₉ acid. The distribution patterns of the acids differed significantly among the algal samples. Hence 2-hydroxy acids may be useful for the classification of algal species as well as being an important source of 2-hydroxy acids in the natural environment.