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.     

Physiological and biochemical contributions to the taxonomy of the genus Chlorella

Starch hydrolysis, i.e., the production of extracellular amylolytic enzymes, was found to be a specific character for most species of the genus Chlorella. C. fusca var. vacuolata, C. spec. 211-30, and C. spec. 211-11r hydrolyse starch, whereas C. vulgaris, C. fusca var. rubescens, C. zofingiensis, C. fusca var. fusca, C. minutissima, C. homosphaera, C. kessleri, C. luteoviridis, and C. protothecoides are unable to hydrolyse starch. Only C. sorokiniana and C. saccharophila appear heterogenous; within C. sorokiniana, 7 strains hydrolyse starch and 9 do not; and within C. saccharophila, 6 strains exhibit amylolytic activity and 2 do not. — A key for the identification, according to 9 easily determined physiological and biochemical characters, of the Chlorella species is presented.     

Upper limits of temperature for growth inChlorella (Chlorophyceae)

The upper limit of temperature for growth is a species-specific character in the genusChlorella. The limits of 14Chlorella species range from 26–30°C (C. saccharophila) to 38–42°C (C. sorokiniana), withC. fusca var.vacuolata (34°C) andC. kessleri (34–36°C) assuming an intermediate position. Thus, there is no wide gap in the temperature limits between the normal (“low-temperature”) species ofChlorella and the “high-temperature” species,C. sorokiniana.     

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.     

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.     

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.     

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.     

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.     

Physiological and biochemical contributions to the taxonomy of the genus Chlorella

1. Rabbit antisera to soluble cytochrome c-553 and to ferredoxin were characterized by double immunodiffusion, the antiserum to cytochrome c-553 also by immuno-electrophoresis and by the quantitative precipitation reaction.
2. Soluble cytochromes c-553 were isolated from 11 strains of Chlorella belonging to 8 species and from one strain of Scenedesmus. Their reactivity with antiserum to soluble cytochrome c-553 from Chlorella fusca var. vacuolata 211-8b was studied.
3. With the double immunodiffusion method, the soluble cytochromes c-553 from C. fusca var. vacuolata 211-8b, C. fusca var. rubescens 232/1, C. fusca var. fusca 343, and Scenedesmus obliquus 276-3b appear immunologically identical.
4. The soluble cytochromes c-553 from C. zofingiensis 211-14a, 211-14b, and 211-14c show in the cross reaction a partial immunological relationship with that from C. fusca var. vacuolata 211-8b.
5. The soluble cytochromes c-553 from C. sorokiniana 211-8k, C. vulgaris 211-11c and 211-19, C. saccharophila 211-1 a, and C. homosphaera 211-8e (Ca.) do not show a cross reaction with that from C. fusca var. vacuolata 211-8b.
6. These results indicate that not only C. fusca var. fusca, but also C. fusca var. vacuolata, C. fusca var. rubescens, and possibly C. zofingiensis might be more closely related to the genus Scenedesmus than to the Chlorella species of the “C. vulgaris group” (i.e., C. sorokiniana, C. vulgaris, and C. saccharophila).
7. An application of the double immunodiffusion method with an antiserum to ferredoxin from C. vulgaris 211-8m revealed that the ferredoxins from 20 strains of 9 Chlorella species and from one strain of Scenedesmus do not exhibit any immunological differences as compared to the homologous antigen.

     

Deoxyribonucleic acid reassociation and interspecies relationships of the genusChlorella (Chlorophyceae)

Phylogenetic relationships within the genusChlorella were studied by means of DNA/DNA hybridization under both optimal and relaxed reassociation conditions as well as by determination of the thermal stability of hybrid DNA duplexes. The results indicate a relationship betweenC. fusca var.fusca, C. fusca var.rubescens, C. fusca var.vacuolata, and the genusScenedesmus. In addition, the strains endosymbiotic withParamecium bursaria seem to be related with theC. vulgaris/sorokiniana group. The relations between most other species, however, could not be sufficiently resolved by the above methods. This implies considerable phylogenetic divergency within the genusChlorella.     

EFFECT OF MALTOSE RELEASE ON UPTAKE AND ASSIMILATION OF AMMONIUM BY SYMBIOTIC CHLORELLA (CHLOROPHYTA)1

When incubated at pH 4–5, Chlorella freshly isolated from symbiosis with Hydra viridissima PALLAS 1766 (green hydra) release large amounts of photosynthetically fixed carbon in the form of maltose, and assimilation of inorganic N is inhibited. Physiological responses to N starvation of the cultured 3N813A strain of maltose-releasing Chlorella differed from those caused by 48 h of maltose release induced by low pH. N starvation increased rates of ammonium assimilation at pH 7.0 in light or darkness, and ammonium assimilation in darkness stimulated cell respiration. In contrast, cells pretreated at pH 5.0 to induce maltose release were unable to take up ammonium at pH 7.0 unless supplied with an external carbon source such as bicarbonate, acetate, or succinate, and rates of uptake were similar to control cells. Freshly isolated symbionts displayed a similar dependency. Rates of ammonium uptake by cells pretreated at pH 5.0 were reduced in darkness and did not stimulate cell respiration. N-starved cells supplied with ammonium also showed a large short-term increase in glutamine pools at the expense of glutamate, as might be expected if large amounts of ammonium were rapidly assimilated via glutamine synthetase/glutamate synthase, whereas after long-term maltose release cells showed only a small increase in glutamine when supplied with ammonium. Furthermore, maltose release caused a fall in pool sizes of a number of amino acids, including glutamine and glutamate, and also caused a decrease in pool sizes of 2-oxoglutarate and phospho-enol-pyruvate, which are required for ammonium assimilation into amino acids. Cells stimulated to synthesize and release maltose may be unable to assimilate ammonium and synthesize amino acids because of diversion of fixed carbon from N metabolism. We estimate that 40–50% affixed C is required for maximal maltose synthesis, whereas up to 30% fixed C is required for ammonium assimilation. These results are discussed in the context of host regulation of symbiotic algal growth.     

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.

     

On the characteristics and taxonomic position of symbiotic Chlorella

Three strains of symbiotic Chlorella (NC64A, CE/76, UTEX-130) could be assigned to C. vulgaris by physiological and DNA-hybridization studies and a fourth symbiotic strain, 3N8/13-1, exhibited characteristics intermediate between C. vulgaris and C. sorokiniana. None of the strains contained detectable sporopollenin or was nutritionally fastidious but the capacity for sugar release (usually maltose) may be a characteristic of all symbiotic Chlorella.     

Physiological,Biochemical, and Molecular Characters for the Taxonomy of the Subgenera of Scenedesmus (Chlorococcales,Chlorophyta)

Biochemical and physiological properties of 16 Scenedesmus species representing the three subgenera Scenedesmus, Acutodesmus, and Desmodesmus are not suitable for species differentiation. All Scenedesmus species studied thus far produce secondary carotenoids, e.g. astaxanthin and canthaxanthin, under nitrogen-deficient conditions. In addition, with the exception of one strain, hydrogenase activity under anaerobic conditions is generally present. Sequence analyses of ribosomal 18S RNAs indicate that the subgenus Desmodesmus is phylogenetically well separated from the other subgenera, whereas the separation of Scenedesmus and Acutodesmus appears less convincing and is dismissed in favour of a single subgenus, Scenedesmus. Three taxa formerly assigned to the genus Chlorella are shown to be unicellular species of the genus Scenedesmus. “Chlorella” fusca var. vacuolata and “C.” fusca var. rubescens, which is closely related to S. obliquus, belong to the Scenedesmus/‘Acutodesmus group. “C.” fusca var. fusca is closely related to S. communis and thus belongs to the subgenus Desmodesmus. Inclusion of Kermatia pupukensis into the genus Scenedesmus is also strongly supported by the RNA data which furthermore indicate a relationship with the subgenus Desmodesmus.     

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.     

Separation ofChlorella ellipsoidea fromC. saccharophila (Chlorophyceae): No growth on mannitol and cadmium sensitivity

This paper presents physiological evidence for the existence as separate taxonomic entities ofChlorella saccharophila andC. ellipsoidea. C. saccharophila is able to utilize mannitol for growth in the dark, whereasC. ellipsoidea is unable to grow on mannitol. In addition,C. ellipsoidea is characterized by an extreme sensitivity towards cadmium.—Among the otherChlorella species, onlyC. luteoviridis is capable of growing on mannitol.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

The genetic relationships of nine strains of Chlorella saccharophila were determined by DNA hybridization techniques. Four strains are closely related to the type strain 211-9a and one strain seems to be moderately related, whereas the taxonomic position of the remaining three strains is not clear. C. saccharophila, like C. sorokiniana, is another species of Chlorella containing strains which are heterogeneous in their overall DNA base sequence and partly also in morphological, biochemical and physiological characters.     

Two aeromonads: Growth of symbionts fromHydra viridis

A large population of bacteria resides in the gastrodermal and ovarian tissue of the freshwater green coelenterateHydra viridis (Ohio, Jubilee, and Carolina strains). The intracellular bacteria are strongly correlated with the presence of symbiotic chlorellae in the animal cells. The bacteria accompany the chlorellae when they are expelled in vesicles during stages of sexual maturation. Two isolates of these bacteria were taken from ruptured bacterial-algal vesicles flushed out of the enteron of surface-sterilzed hydras. They were cultured on proteose peptone. Both were identified asAeromonas punctata, on the basis of over forty traits. These large, Gram-negative rods differ very little from published characteristics ofA. punctata subsp.punctata. UnlikeA. punctata subsp.punctata, the hydra symbionts grew on KCN broth and lacked the lysine decarboxylase reaction. We identified the two isolates as members of the same new strain ofAeromonas punctata symbiotic inHydra viridis. A different aeromonad could not be isolated from vesicles flushed out of surface-sterilized hydras, but it appeared along with the first aeromonad on plates from which smears of the holdfast and hypostome region of ethanol-rinsed hydra were made. This second orange-pigmented bacterium in a member of the holdfast microbial community associated with hydra and hydra eggs. It differed fromAeromonas hydrophila subsp.anaerogenes in only 5 out of over 40 traits tested. All differences were losses of metabolic activities. Both hydra-associatedA. hydrophila andA. punctata, which are easily grown, and yet form regular and natural associations with the greenHydra viridis, may prove useful for understanding metabolic relationships in micro-organisms adapted for symbiotic associations.     

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     

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.