The Occurrence of the Glucose-inducible Transport Systems for Glucose,Proline, and Arginine in Different Species of Chlorella

Incubation of the green alga Chlorella vulgaris (strain K, Tanner and Kandler, 1967) with glucose leads to the induction of a glucose transport system and of two amino acid transport systems. Because it was not clear whether the regulation of 3 different transport systems by glucose is specific to our strain of Chlorella or whether it is a general property of the genus Chlorella, 11 other free living and symbiotic Chlorella species and strains were tested for glucose-inducible glucose, arginine and proline transport. It was found that nearly all Chlorella species possess glucose and amino acid uptake systems. Often they were constitutive, although in some species they were induced or stimulated by glucose. According to the transport activities of the different Chlorella species and strains, a physiological classification of Chlorella was constructed, resulting in 3 groups: the C. fusca vacuolata, the C. vulgaris and the symbiotic Chlorella group. Our Chlorella (strain K) obviously belongs to the C. vulgaris group and forms a link to symbiotic Chlorella strains. This suggests that the possession of the glucose-regulated transport systems is of advantage for Chlorella in symbiotic situations, whereas the constitutive systems are useful for free living Chlorella.     

Comparative studies on photosynthetic enzymes of the symbiotic Chlorella from Paramecium bursaria and other symbiotic and non-symbiotic Chlorella strains

The activities of ribulose 1,5-bisphosphate carboxylase and of carbonic anhydrase were studied in cell-free extracts of two symbiotic Chlorella strains isolated from Paramecium bursaria and from Spongilla sp., and of two nonsymbiotic strains of Chlorella (Chlorella fusca and Chlorella vulgaris) cultivated at varied CO₂-concentrations. The symbiotic Chlorella of Paramecium bursaria differs distinctly from the other Chlorella strains by a higher activity of ribulose 1,5-bisphosphate carboxylase, which is independent of the actual CO₂-concentration, and by a lack of carbonic anhydrase activity. These properties are discussed with respect to their ecological significance.Abbreviations CA carbonic anhydrase - Pbi Paramecium bursaria isolate - RuBP ribulose 1,5-bisphosphate Dedicated to Prof. Dr. André Pirson on the occasion of his 70th birthday     

Comparative studies onChlorella cell walls: Induction of protoplast formation

Among 12 strains ofChlorella ellipsoidea, C. vulgaris, andC. saccharophila tested, 4 strains (1,C. ellpsoidea; 2,C. vulgaris; 1,C. saccharophila) formed osmotically labile protoplasts after treatment with mixtures of polysaccharide degrading enzymes. The relationship between enzymatical digestibility and structure or composition ofChlorella cell walls were studied by electron microscopy and staining techniques with some specific dyes. The cell wall structures of the 12Chlorella strains were grouped into three types: (1) with a trilaminar outer layer, (2) with a thin outer monolayer, and (3) without an outer layer. Protoplasts were formed only from the strains with a cell wall of Type 2. In the strains with a cell wall of Type 1, the outer layer protected the inner major microfibrillar layer against enzymatic digestion. The cell wall of Type 3 was totally resistant to the enzymes; the chemical composition of the cell wall would be somewhat different from that of other types.     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Excretion of Sugars by Chlorella Species Capable and Incapable of Symbiosis with Hydra viridis

The excretion of some sugars (maltose, glucose, and glucose-6-phosphate) was studied at pH 2.5–6.0 in 38 strains of Chlorella belonging to 15 species of which 7 are capable and 8 incapable of symbiosis with Hydra viridis. A high rate of maltose excretion below pH 4.0 (Cernichiari et al., 1969) was found only in C. vulgaris (non-symbiotic) and C. mirabilis (non-symbiotic). The other Chlorella species are characterized by quite different patterns of sugar excretion. C. spec. (= “C. paramecii”; symbiotic) excretes very high amounts of maltose in the whole range from pH 2.5–6.0. C. kessleri (symbiotic), C. luteoviridis (symbiotic), and C. fusca var. fusca (non-symbiotic) show a predominant excretion of glucose-6-phosphate from pH 2.5–6.0. Some strains also exhibit a high excretion of glucose above pH 4.0 (C. spec. = “C. paramecii”) or below pH 3.0 (C. fusca var. vacuolata). Several species, e.g. C. saccharophila var. saccharophila (symbiotic), C. sorokiniana (non-symbiotic), and C. protothecoides (symbiotic), excrete only very small amounts of sugars. There is no obvious correlation between sugar excretion and the ability or inability of the Chlorella species to form stable symbioses with Hydra viridis.     

Species-specific ability ofChlorella strains (Chlorophyceae) to form stable symbioses withHydra viridis

46 strains ofChlorella, identified by physiological and biochemical characters, were examined for their ability to form stable symbioses with aposymbioticHydra viridis. It was found to be a species-specific characteristic. Among the 15 taxa studied, onlyC. saccharophila var.ellipsoidea, C. saccharophila var.saccharophila, C. fusca var.vacuolata, C. kessleri, C. luteoviridis, andC. protothecoides formed stable symbioses withHydra viridis. Among the 11 known physiological and biochemical characters of theseChlorella species, only acid tolerance seems to be correlated with symbiosis: All symbiotic species are capable of growing at or below pH 4.0.     

Zur Taxonomie einer auxotrophen Chlorella aus Paramecium bursaria Ehrbg

Zusammenfassung Auf Grund der physiologischen Merkmale einer in Paramecium bursaria Ehrbg. auftretenden Chlorella ergibt sich eine systematische Zuordnung in den Formenkreis um Chlorella vulgaris f. tertia Fott et Nováková und Chlorella vulgaris var. vulgaris Beijerinck. Hiervon abweichende Befunde anderer Autoren werden diskutiert.

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On the taxonomy of an auxotrophic Chlorella isolated from Paramecium bursaria ehrbg

An auxotrophic Chlorella has been isolated from Paramecium bursaria Ehrbg. and cultivated in mass culture in an inorganic medium supplied with vitamins B₁ and B₁₂. With regard to its physiological properties it is not identical with either one of the so far known Chlorella species. It belongs, however, to the group of Chlorella vulgaris f. tertia Fott et Novaková and Chlorella vulgaris var. vulgaris Beijerinck.

     

Physiologische und biochemische Beiträge zur Taxonomie der Gattung Chlorella

Zusammenfassung Es wurde die Verwendbarkeit von Acetat, Glucose, Fructose, Galactose, Saccharose und Lactose als Kohlenstoffquelle für das Wachstum von 72 Chlorella-Stämmen, die 10 autotrophen Taxa angehören, untersucht. Im Dunkeln zeigen mit Acetat 34 Stämme und mit Glucose 37 Stämme gutes Wachstum (Chlorella kessleri, die meisten Stämme von C. vulgaris und C. vulgaris f. tertia, sowie einige wenige Stämme von C. fusca), während Fructose von 21 Stämmen verwertet wird (C. kessleri, die meisten Stämme von C. luteoviridis und C. saccharophila, sowie einige Stämme von C. fusca und C. zofingiensis). Gutes Wachstum mit Galactose wurde bei 11 Stämmen gefunden (C. kessleri und einige Stämme von C. vulgaris). Saccharose und Lactose ermöglichen dagegen kein intensives Wachstum. Die Verwendbarkeit der 6 geprüften organischen Verbindungen für heterotrophes Wachstum ist als taxonomisches Merkmal zur Charakterisierung von Chlorella-Arten nicht geeignet. Lediglich Chlorella kessleri Fott et Nováková, die allgemein die ausgeprägteste Fähigkeit zu heterotrophem Wachstum besitzt, unterscheidet sich durch gute Verwertung von Galactose von den übrigen Arten.

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Physiological and biochemical contributions to the taxonomy of the genus Chlorella VI. Utilization of organic carbon compounds

Summary The utilization of acetate, glucose, fructose, glactose, saccharose, and lactose as sources of carbon for growth in the dark of 72 Chlorella strains belonging to 10 autotrophic taxa was studied. 34 strains grow well with acetate and 37 strains with glucose (Chlorella kessleri, most strains of C. vulgaris and C. vulgaris f. tertia, and a few strains of C. fusca), and fructose is utilized by 21 strains (C. kessleri, most strains of C. luteoviridis and C. saccharophila, and some strains of C. fusca and C. zofingiensis). 11 strains show good growth with galactose (C. kessleri and some strains of C. vulgaris). Saccharose and lactose, on the other hand, do not support vigorous growth. Utilization of the 6 organic compounds cannot serve as a taxonomic character in the genus Chlorella. However, Chlorella kessleri Fott et Nováková shows the most pronounced ability for heterotrophic growth and differs in its good growth with galactose from the other species.

     

Molecular characterization of Chlorella cultures of the National Institute for Environmental Studies culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov., and Didymogenes soliella sp. nov. (Chlorellaceae,Trebouxiophyceae)

Chlorella Beijerinck (Chlorellaceae, Trebouxiophyceae) strains from the collection of the National Institute for Environmental Studies (NIES) were characterized using gene sequence data. The misidentification of a number of strains was rectified. Chlorella vulgaris Beijerinck NIES‐2173 was reclassified as C. sorokiniana Shihira et Krauss. Chlorella sp. NIES‐2171 was described as a new species in the genus Micractinium Fresinius, M. inermum Hoshina et Fujiwara. Chlorella sorokiniana NIES‐2167 and Chlorella sp. NIES‐2330 were found to be phylogenetically related to Didymogenes Schmidle. We propose these two strains be transferred to the genus Didymogenes and given new names: D. sphaerica Hoshina et Fujiwara and D. soliella Hoshina et Fujiwara. Taxonomic decisions were primarily based on small subunit‐internal transcribed spacer ribosomal DNA phylogeny for genus assignment and ITS2 sequence‐structure to determine species autonomy. Our findings suggest that this strategy is the most effective way to use the species concept among autosporic coccoids.     

Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar phaseoli isolated from soils of Rwanda

We have isolated 48 strains of Rhizobium leguminosarum biovar phaseoli from nodules of Phaseolus vulgaris L. cultivated on 32 different soils at 22 various locations in Rwanda, Central Africa. The symbiotic effectiveness of the strains was appraised in the greenhouse by measuring shoots dry matter and total plant nitrogen content after six weeks of growth. Of the strains tested 19%, 58% and 23% were rated very effective, effective and ineffective, respectively. A very significant correlation (r=0.96, P<0.01) was observed between shoots dry matter and total N content. By using the total nitrogen balance method, it was estimated that in the presence of a very effective strain, up to 86% of the N present in the shoots comes from N₂ fixation. No significant correlations were observed between the symbiotic effectiveness of the strains and the pH of the soils from which they originated, the tolerance of the strains to acidity or their ability to produce organic acids. The nine very effective strains selected were highly competitive against two ineffective strains with the two P. vulgaris cultivars Rubona-5 and Kiryumukwe.Contribution no 367, Station de recherches, Agriculture Canada.Contribution no 367, Station de recherches, Agriculture Canada.     

Taxonomic implications of Prototheca and Chlorella cell wall polysaccharide characterization

Summary Four polysaccharide fractions were obtained by acid and alkaline degradation of purified cell walls of Prototheca spp. and Chlorella spp. These fractions were further hydrolyzed and the component sugars identified. Five Prototheca strains and two Chlorella protothecoides strains have essentially similar polysaccharide compositions, which significantly differ from those of C. vulgaris and C. pyrenoidosa. This emphasizes the close affinity of C. protothecoides to Prototheca spp. not shared by other Chlorella spp.     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.     

Photobehaviour of Paramecium bursaria infected with different symbiotic and aposymbiotic species of Chlorella

The endosymbiotic unit of Paramecium bursaria and Chlorella spec. shows two types of photobehaviour: 1) A step-up photophobic response which possibly depends on photosensitive agents in the ciliate cell itself — as is also shown by alga-free Paramecium bursaria - and can be drastically enhanced by photosynthetic activity of symbiotic algae; and 2) a step-down photophobic response. The step-down response leads to photoaccumulation of green paramecia. Both types of photobehaviour in Paramecium bursaria do not depend on any special kind of algal partners: The infection of alga-free Paramecium bursaria with different Chlorella species results in new ciliatealgae-associations. They are formed not only by combination of the original symbiotic algae with their host, but also by infection with other symbiotic or free-living (aposymbiotic) chlorellae, respecitively. Systems with other than the original algae are not permanently stable — algae are lost under stress conditions — but show the same types of photobehaviour. Photoaccumulation in general requires algal photosynthesis and occurs only with ciliates containing more than fifty algae/cell. It is not mediated by a chemotactic response to oxygen in the medium, since it occurs at light fluence rates not sufficient for a release of oxygen by the symbiotic system, e.g., below its photosynthetic compensation point. Photoresponses can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Sensory transduction does not depend on any special symbiotic features of the algae, e.g., sugar excretion. The participation of oxygen in the Paramecium cell, of its cytoplasmic pH and of ions released or taken up by endosymbiotic algae in sensory transduction is discussed.     

SUGAR COMPOSITION OF THE CELL WALL AND THE TAXONOMY OF CHLORELLA (CHLOROPHYCEAE)1

Cell walls of forty Chlorella strains covering all species of the Algal Collection of Göttingen (C. fusca var. vacuolata, C. kessleri, C. luteoviridis, C. minutissima, C. protothecoides, C. saccharophila, C. sorokiniana, C. vulgaris, and C. zofingiensis) were compared. The nine species were divided into two groups according to the major sugar in the rigid wall. The first group had a glucose-mannose-rigid wall and included C. fusca var. vacuolata, C. luteoviridis, C. minutissima, C. protothecoides, C. saccharophila, and C. zofingiensis. The second group, with a glucosamine-rigid wall, included C. kessleri, C. sorokiniana, and C. vulgaris. Chlorella strains of the nine species were further classified by constituent sugars, ruthenium red stainability, and anisotropy of the cell walls.     

Glucose uptake by symbiotic Chlorella in the green-hydra symbiosis

There is a correlation between the ability of symbiotic Chlorella algae to take up glucose and their survival in green hydra grown in continuous darkness. Although normal symbionts of European green hydra, which persist at a stable level in dark-grown animals, possessed no detectable constitutive ability to take up glucose when grown in light, uptake was induced after incubation in a medium containing glucose. Further, symbionts isolated from hydra grown in darkness for two weeks had acquired a constitutive uptake ability. Neither NC64A nor 3N813A strains of algae, in artificial symbiosis with hydra, persisted in dark-grown animals, and they showed little or no uptake ability, although in culture NC64A possessed both constitutive and inducible glucose-uptake mechanisms. In contrast, mitotic indices in all three types of algae in symbiosis with hydra increased after host feeding, indicating that the factor which stimulates algal cell division is not identical to the substrate utilised during heterotrophic growth.Abbreviations E/E normal Hydra-Chlorella symbiosis - E/NC, E/3N artificial symbioses between Hydra and Chlorella strains NC64A and 3N813A, respectively - 3-OMG 3-O-methyl-D-glucose - SDS sodium dodecyl sulfate     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA hybridization techniques showed Chlorella fusca var. vacuolata and C. kessleri to be homogeneous species with DNA homologies of 90–100% C. fusca var. fusca and var. rubescens, however, have only about 15% DNA homology with C. fusca var. vacuolata and should no longer be regarded as varieties. A good correlation was found so far between biochemical and physiological characters used in the taxonomy of Chlorella and DNA relatedness. Mutant strains of Chlorella were tested for DNA homologies to prove the reliability of the taxonomical interpretation.     

大型海藻龙须菜抽提液对褶皱臂尾轮虫生命表参数的影响

大型海藻富含多种活性物质,具有抗衰老等生物活性;轮虫是良好的潜在抗衰老研究模式生物。本研究以褶皱臂尾轮虫(Brachionus plicatilis)作为实验对象,研究了不同浓度的大型海藻龙须菜抽提液(0,250,500,750,1000 mg/L)和不同浓度的食物(蛋白核小球藻和普通小球藻)对褶皱臂尾轮虫生命表参数的影响。结果表明:与对照组相比,食物浓度为1.0×10~6个/mL蛋白核小球藻时,不同浓度龙须菜抽提液对轮虫产卵数、平均寿命、净生长率以及世代时间有显著促进效应(P0.05);轮虫平均产卵数及寿命在龙须菜抽提液浓度750 mg/L处达到最高,分别为16只和13.9d(P0.05)。食物浓度为2.0×10~6个/mL普通小球藻时,轮虫平均产卵数和寿命在抽提液浓度为500 mg/L处达到最高,分别为16只和13.6d(P0.05),轮虫平均寿命和净生长率均有显著提高(P0.05)。相同龙须菜抽提液浓度下,食物浓度为1.0×10~6个/mL蛋白核小球藻下轮虫的净生长率、世代时间均显著高于食物浓度为2.0×10~6个/mL蛋白核小球藻培养的轮虫(P0.05);食物浓度为2.0×10~6个/mL时,普通小球藻培养轮虫的净生长率和世代时间均显著高于蛋白核小球藻实验组(P0.05)。交互作用分析显示,龙须菜抽提液与小球藻的交互作用对褶皱臂尾轮虫的内禀增长率有显著影响(P0.05)。研究结果表明,大型海藻龙须菜抽提液对褶皱臂尾轮虫的生长与生殖有促进作用,延长轮虫寿命。     

The endosymbiotic unit ofStentor polymorphus andChlorella sp. morphological and physiological studies

Summary The greenStentor polymorphus harbours unicellular coccoid chlorophycean algae. They are located in food vacuoles, where they show various states of digestion, as well as in individual so-called perialgal vacuoles. According to their characteristic morphological properties the algae belong to the genusChlorella. They can be isolated from the ciliate and cultivated in mass cultures in a sterile defined inorganic medium supplemented with vitamins B₁ and B₁₂. The algae have no secondary carotenoids and excrete maltose by a pH-dependent mechanism. They thus show a conspicuous physiological similarity to the symbiotic chlorellae ofParamecium bursaria andHydra viridis, which also excrete maltose.A comparison of the properties of the chlorellae isolated fromStentor polymorphus and of the intactStentor polymorphus-Chlorella unit with the characteristic features of symbiotic chlorellae and with endosymbiotic systems containingChlorella sp. in general, lead to the conclusion that the greenStentor polymorphus is also a true endosymbiotic system.     

Biochemical Taxonomy of Symbiotic Chlorella Strains from Paramecium and Acanthocystis*

Biochemical and physiological characters of 5 symbiotic Chlorella strains, 4 of them from Paramecium (Ciliata) and one from Acanthocystis (Rhizopoda, Heliozoa), were studied. Four strains (3 from Paramecium and one from Acanthocystis) belong to the same species. This is characterized by the presence of hydrogenase, no formation of secondary carotenoids, no growth on mannitol, requirements for thiamine and vitamin B₁₂, and a G + C content of the DNA of 66.4–68.4 mol%; the limits of growth are at pH 5.5, at up to 1% NaCl, and at 26–30°C. The strains are somewhat heterogeneous in their utilization of inorganic nitrogen sources: only two of them are able to use nitrate, whereas all can grow with nitrite and ammonium. Thus, in two strains the nitrate-reducing system — in contrast to nitrite reductase — seems to be defective. Another strain, which has been claimed to be from Paramecium, belongs to C. protothecoides.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.