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     

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.

     

Evidence of de novo synthesis of maltose excreted by the endosymbiotic Chlorella from Paramecium bursaria

The endosymbiotic Chlorella sp. from Paramecium bursaria excretes maltose both in the light and in the dark. Experiments on photosynthetic ¹⁴CO₂ fixation and ¹⁴CO₂ pulse-chase experiments show that maltose is synthesized in the light directly from compounds of the Calvin cycle, whereas in the dark it results from starch degradation.     

Die stoffwechselphysiologischen Benziehungen Zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose

Zusammenfassung Der endosymbiontische Verband von Paramecium bursaria Ehrbg. mit Chlorella spec. (grünes Paramecium) wurde physiologisch und cytologisch untersucht. Ein Vergleich der Eigenschaften der Symbiosecinheit mit denen der getrennt kultivierten Symbiosepartner ergab die folgenden Merkmale und Unterschiede: 1. Der symbiontische Verband hat bis zu einer Beleuchtungsstärke von 6000 lux eine stärkere Photosyntheseleistung als die aus ihm isolierte und in Massenkultur in einem definierten Medium kultivierte Alge. Algenfreie P. bursaria zeigen nur eine minimale Fähigkeit zur CO₂-Fixierung. 2. Der Kompensationspunkt der Photosynthese liegt beim algenhaltigen Paramecium bei ca. 4000–5000 lux, derjenige der getrennt kultivierten Alge bei ca. 200–400 lux. 3. Die Symbioseeinheit hat im Dunkeln im Vergleich mit algenfreien P. bursaria einen niedrigeren, im Vergleich mit der frei kultivierten Alge jedoch einen höheren Sauerstoffbedarf. 4. Das grüne Paramecium nimmt weniger Kohlenhydrate aus dem Medium auf als algenfreie Paramecien, hat aber eine höhere Aufnahmeleistung als die isoliert gezogenen Algen. 5. Im Symbioseverband besitzt die symbiontische Alge im Licht eine kompakte Lagerung der photosynthetischen Membranen und eine massive Stärkeablagerung. Die Vergiftung der Photosynthese durch 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) oder die Kultur im Dunkeln führt in algenhaltigen Paramecien zu einer aufgelockerten Lagerung der Thylakoide und einer Verringerung der Stärkeablagerung. Die Algen-population unterliegt im symbiontischen Verband einem komplexen Regulationsmechanismus, bei dem u. a. der intracelluläre Kohlenhydratspiegel eine Rolle spielt. Die geschilderten Ergebnisse werden im Zusammenhang mit der Ökologie des grünen P. bursaria diskutiert.

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The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosisII. Symbiosis-specific properties of the physiology and the cytology of the symbiotic unit and their regulation

The endosymbiotic association of Paramecium bursaria Ehrbg. with Chlorella spec. (green Paramechim) was studied both physiologically and cytologically. Comparison of the properties of the symbiotic unit with those of the symbiotic partiners which bad been isolated from it revealed the following features and differences: 1. Up to 6000 lux the photosynthetic capacity of the symbiotic unit is higher than that of the isolated symbiotic algae grown independently in mass culture under defined conditions. Alga-free. Paramecium bursaria (colourless Paramecium) show a very low rate of CO₂-fixation. 2. The green Paramecium has a higher compensationpoint of photosynthesis (4000–5000 lux) than the isolated alga (200–400 lux). 3. Green paramecia consume less oxygen in darkness than colourless organisms but more than the isolated algae. 4. The uptake of carbohydrates from the culture medium by green paramecia is lower than the uptake by alga-free P. bursaria but higher than the one of the isolated algae. 5. Symbiotic algae within the intact symbiotic unit show tightly packed photosynthetic membranes and an intense deposition of starch. In the presence of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) or in darkness the arrangement of thylakoids is less compact and the deposition of starch is reduced. The growth and the number of the symbiotic algae in situ is regulated by a complex mechanism to which the intracellular level of carbohydrates belongs. The results are discussed in connection with ecological aspects of the Paramecium bursaria-endosymbiosis.

     

Die stoffwechselphysiologischen Beziehungen zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose

Zusammenfassung Infektionsexperimente algenfreier Paramecium bursaria mit aus diesen isolierten und unter Stickstoffmangel-Bedingungen vorkultivierten Algen deuten darauf hin, daß die Versorgung der endosymbiontischen Algen mit stickstoffhaltigen Verbindungen durch ihren Wirt in einem zu gutem Wachstum und Vermehrung der Alge ausreichendem Maße möglich ist. Die Bedeutung dieser stoffwechselphysiologischen Beziehung für die Symbiosepartner wird diskutiert.Die Vergiftung der Photosynthese der endosymbiontischen Chlorella durch 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) führt in grünen Paramecium bursaria durch Beeinflussung des Kohlenstoff-Stoffwechsels zu einer Entkoppelung des symbiontischen steady state-Systems und damit zur Auflösung der Symbiose. Eine ausreichende heterotrophe Ernährung der Alge durch das Paramecium ist in der Symbiose offenbar nicht möglich.Die Anwendung von 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) kann als neue Methode zur Züchtung algenfreier Paramecium bursaria dienen.

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The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosisI. The nitrogen and the carbon metabolism

Symbiotic Chlorellae have been isolated from Paramecium bursaria Ehrbg. and cultivated under conditions of nitrogen deficiency. Reinfection of Chlorella-free Paramecium bursaria with these nitrogen-deficient algae resulted in a complete regeneration and multiplication of the algae within the host cells. The endosymbiotic algal cells of the Paramecium bursaria-symbiosis can be supplied by their host with nitrogen.The inhibition of photosynthesis by 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) leads in green Paramecium bursaria to a breakdown of the symbiotic steady state-system resulting in a loss of algal cells. Obviously the endosymbiotic algae cannot be fed heterotrophically by their host to such an extent that a stable symbiosis is maintained.The application of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) can be used as a new method for culturing Chlorella-free Paramecium bursaria.

     

Identification of Chlorella viruses in Paramecium bursaria clones by pulse-field electrophoresis

The ciliates Paramecium bursaria contain endosymbiotic green algae Chlorella spp. in their cytoplasm. The algae isolated from P. bursaria are sensitive to large DNA-containing viruses of the family Phycodnaviridae. The type virus of this family is PBCV-1 (Paramecium bursaria Chlorella virus). Investigation of the total DNA of P. bursaria clones by pulse-field electrophoresis (PEGE) revealed a pronounced band on PEGE profiles of some P. bursaria clones; the band was formed by DNA molecules of approx. 300 kb. This band probably contained the DNA of Chlorella virus. Two approaches were used in the present work to confirm this hypothesis. Microbiological tests were used to scan a collection of P. bursaria clones for specific types of viruses; the 300-kb band was revealed only in the PEGE profiles of virus-containing clones. Blot hybridization of P. bursaria total DNA separated by pulse-field electrophoresis with the virus-specific probe revealed that the band under study was formed by the DNA of a Chlorella virus. Paramecium clones were shown to contain approx. 10⁵ copies of nonintegrated viral DNA.     

The role of endosymbiotic algae in photoaccumulation of green Paramecium bursaria

The endosymbiotic unit of Paramecium bursaria with Chlorella sp. photoaccumulates in white, blue-green, and red light (<700 nm), whereas alga-free Paramecia never do. The intensity of photoaccumulation depends on both the light fluence rate and the size of the symbiotic algal population. Photoaccumulation can be stopped completely with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. Hence the photosynthetic pigments of the algae act as receptors of the light stimulus for photomovement and a close connection must exist between photosynthesis of the algae and ciliary beating of the Paramecium.     

Cloning and characterization of endosymbiotic algae isolated fromParamecium bursaria

Summary The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 10⁷ algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 10³ algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

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.     

Correlation between circadian periods and cellular activities in Paramecium bursaria

Paramecium bursaria shows a circadian rhythm of photoaccumulation: photoaccumulation is stronger during the day than at night. We obtained five strains of P. bursaria having different circadian periods under continuous light conditions, ranging from 20.9 to 27.9 h. Various physiological activities were compared in the cells of these strains. The periods of contractile vacuole contraction were in the range 10–15 s, which was almost proportional to the periods of the circadian rhythm in each strain. Swimming velocities were inversely proportional to the circadian period; i.e. swimming velocities were high in strains whose circadian periods were short. Resting membrane potential was more depolarized in strains with longer circadian periods. Finally, the membrane resistance of the resting state was reduced in proportion to the increase of the circadian period. Such correlation between the cellular properties and the circadian period suggests that the circadian clock mechanism is associated with various physiological activities of the cell.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

Photosynthesis dependent acidification of perialgal vacuoles in theParamedum bursaria/Chlorella symbiosis: Visualization by monensin

Summary After treatment with the carboxylic ionophore monensin theChlorella containing perialgal vacuoles of the greenParamecium bursaria swell. TheParamecium cells remain motile at this concentration for at least one day. The swelling is only observed in illuminated cells and can be inhibited by DCMU. We assume that during photosynthesis the perialgal vacuoles are acidified and that monensin exchanges H⁺ ions against monovalent cations (here K⁺). In consequence the osmotic value of the vacuoles increases. The proton gradient is believed to drive the transport of maltose from the symbiont into the host. Another but light independent effect of the monensin treatment is the swelling of peripheral alveoles of the ciliates, likewise indicating that the alveolar membrane contains an active proton pump.Abbreviations HEPES N-(2-hydroxyethyl)piperazine-N-2-ethane sulfonic acid - DCMU 3-(3, 4-dichlorophenyl)-1,1-dimethylurea     

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding.     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.     

Photo-oxidative stress in symbiotic and aposymbiotic strains of the ciliate Paramecium bursaria.

We tested the hypothesis that photo-oxidative stress is greater in symbiotic representatives of the freshwater ciliate Paramecium bursaria than in aposymbiotic (i.e., without Chlorella) ones. The level of oxidative stress was determined by assessing reactive oxygen species (ROS) with two fluorescent probes (hydroethidine and dihydrorhodamine123) by flow cytometry in exponential and stationary growth phases of both strains. Photo-oxidative stress was assessed in the laboratory after exposure of the ciliates to photosynthetically active radiation (PAR: 400-700 nm) and PAR+ultraviolet radiation (UVR: 280-400 nm). Additionally, both strains were screened for their antioxidant defenses by measuring the activity of the enzymes catalase, superoxide dismutase (SOD), and glutathione reductase. The results showed that aposymbiotic ciliates had higher levels of PAR-induced oxidative stress than symbiotic ones. Significant differences in PAR-induced oxidative stress were also found in both strains when comparing exponential and stationary growth phases with generally higher values in the former. After exposure to UVR, aposymbiotic ciliates in the stationary phase had the highest levels of ROS despite an increase in SOD activity. By contrast, exposure to UVR decreased catalase activity in both strains. Overall, our results suggest that in this ciliate symbiosis, the presence of symbionts minimizes photo-oxidative stress. This work represents the first assessment of photo-oxidative stress in an algal-ciliate mutualistic symbiosis.