Euglena Cells Research Articles

Euglena gracilis Ribonucleotide Reductase: THE EUKARYOTE CLASS II ENZYME AND THE POSSIBLE ANTIQUITY OF EUKARYOTE B12 DEPENDENCE

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.

Behavior of mitochondria, chloroplast and pyrenoid in synchronized cells of Chlamydomonas and Euglena

Summary: Giant mitochondria are temporarily formed, probably by fusion of smaller mitochondria, in Chlamydomonas reinhardi cells at an intermediate stage in the growth phase of the cell cycle. The formation of giant mitochondria is accompanied by a marked decrease in the oxygen-uptake activity of the cells and the division of giant mitochondria into smaller forms by a re-increase in the activity. Changes in the morphology of mitochondria of Euglena gracilis Z cells were also followed with an electron microscope during the cell cycle in a synchronous culture under photoautotrophic conditions. Giant mitochondria were temporarily formed, most probably by fusion of smaller forms, in the cells at an intermediate stage in the growth phase of the cell cycle. Furthermore, we found for the first time such phenomena in chloroplasts of Euglena gracilis Z, called?ggiant chloroplast?h, and we clarified their arrangement in the cell cycle. Changes in morphology of the pyrenoid and those in distribution of RuBisCO in chloroplasts were followed by immunoelectron microscopy during the growth and division phases of synchronized cells of Euglena.The immuno-reactive protein were densely localized in the pyrenoid, and thinly distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. From a comparison of photosynthetic CO2-fixation with the total carboxylase activity of RuBisCO extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO2-fixation in photosynthesis.

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (km=68 microm) and trans-2-hexenoyl-CoA (km=91 microm). In the crotonyl-CoA-dependent reaction, both NADH (km=109 microm) or NADPH (km=119 microm) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function.

Immunogold Localization of Photosynthetic Proteins in Euglena

Summary: Changes in pyrenoid morphology and the distribution within the chloroplast of ribulose-1, 5-bisphosphate carboxylase/oxygenase(RuBisCO)of Euglena gracilis Z were followed by immunoelectron microscopy during growth and division phase. Most of the immunoreactive protein was found in the pyrenoid during the growth phase with only a small amount of gold particles localized to the stroma. During the division phase, the pyrenoid was not detected and the gold particles were dispersed throughout the stroma. A comparison between rates of photosynthetic CO2-fixation and the amount of total carboxylase activity catalyzed by RuBisCO extracted from Euglena cells in the growth phase suggests that pyrenoid localized RuBisCO is responsible for photosynthetic CO2-fixation. The precursors to the Euglena light-harvesting chlorophyll a/b binding proteins of photosystem II(LHCP II)are large polyproteins containing multiple copies of LHCP II covalently joined by a conserved decapeptide. Light induced LHCP II synthesis is controlled at the translational level in Euglena rather than the transcriptional level as found in higher plants and other algae. Under conditions promoting LHCP II synthesis and accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed by immunogold electron microscopy in the Golgi apparatus. The kinetics of LHCP II appearance in the Golgi apparatus as measured by immunogold electron microscopy in synchronous cells and by pulse labeled with H2[35S]O4 in cells undergoing normal light-induced chloroplast development suggests that nascent LHCP II is transported to the Golgi apparatus prior to chloroplast import and thylakoid insertion.

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.

Effects of temperature, CO 2/O 2 concentrations and light intensity on cellular multiplication of microalgae, Euglena gracilis

Microalgae culture is likely to play an important role in aquatic food production modules in bioregenerative systems for producing feeds for fish, converting CO 2 to O 2 and remedying water quality as well as aquatic higher plants. In the present study, the effects of culture conditions on the cellular multiplication of microalgae, Euglena gracilis, was investigated as a fundamental study to determine the optimum culture conditions for microalgae production in aquatic food production modules including both microalgae culture and fish culture systems. E. gracilis was cultured under conditions with five levels of temperatures (25–33 °C), three levels of CO 2 concentrations (2–6%), five levels of O 2 concentrations (10–30%), and six levels of photosynthetic photon flux (20–200 μmol m −2 s −1). The number of Euglena cells in a certain volume of solution was monitored with a microscope under each environmental condition. The multiplication rate of the cells was highest at temperatures of 27–31 °C, CO 2 concentration of 4%, O 2 concentration of 20% and photosynthetic photon flux of about 100 μmol m −2 s −1. The results demonstrate that E. gracilis could efficiently produce biomass and convert CO 2 to O 2 under relatively low light intensities in aquatic food production modules.

The molecular biology of Euglena gracilis. XV. Recovery from centrifugation-induced stratification.

The contents of Euglena gracilis cells can be separated in vivo by ultracentrifugation. Within the unbroken cell, each set of components forms a distinct layer according to their respective densities. The degree of segregation increases with both the g-force and the time of centrifugation, up to a maximum at 100,000 x g for 1 h, when six distinct strata can be observed. When returned to normal growth conditions, essentially all the cells return to the normal state and growth pattern. Greater g-forces or longer exposures do not alter the observable strata, but the ability of the cells to recover is diminished. Smaller g-forces result in less separation of cellular contents and all cells recover, even after 18 h of exposure. Euglena cells stratified at 100,000 x g for 1 h were returned to normal growth conditions; recovery was followed microscopically and by the rate of utilization of oxygen as well as that of the single carbon source. The cells recovered their normal state within 1 to 2 h, which is only a tenth of the normal doubling time. The mechanism for this recovery involves a natural process of change in cell shape caused by contraction and relaxation of the pellicle, a cell surface structure.

Effect of iron on the expression of ascorbate peroxidase in Euglena gracilis

We have investigated the effect of Fe on the induction of ascorbate peroxidase (APX) in Fe-deficient Euglena cells that completely lacked APX activity. Addition of 0.13 mM Fe to Fe-deficient cells caused a two-phase induction of the APX activity: a sharp rapid increase in the first 3 h, followed by a steady gradual increase from 3 through 24 h. Cycloheximide had no effect on the initial increase of activity, but completely inhibited the subsequent increase. Immunoblot analyses using a monoclonal antibody (EAP1) raised against Euglena APX indicated that the former increase is due to activation of APX apo-protein and the latter one is due to de novo synthesis of the enzyme protein. However, Northern analysis revealed that the levels of APX transcripts remained constant, suggesting that the induction of Euglena APX by Fe is regulated at the post-transcriptional stage. The second increase of APX activity was also suppressed by the addition of succinyl acetone, an inhibitor of heme synthesis, suggesting that heme synthesis is essential for the synthesis of APX protein.

Post-transcriptional regulation of ascorbate peroxidase during light adaptation of Euglena gracilis

When Euglena cells are grown heterotrophically in the dark, the chloroplast development stops at the proplastid stage. Upon exposure to light, the cells rapidly develop chloroplasts and the photosynthesis resumes. Here, we have investigated the regulation of cytosolic ascorbate peroxidase (APX) in Euglena gracilis during light adaptation of dark-grown cells. Both the activity and protein levels of APX increased by nearly fourfold in about 24 h of illumination. Northern hybridization with a partial cDNA of Euglena APX as the probe revealed a constant level of APX transcripts during the light adaptation. Similarly, cycloheximide almost completely inhibited APX induction whereas transcription inhibitors did not have a significant effect, suggesting that the light induction of APX is post-transcriptionally regulated. APX induction was abolished when the development of chloroplast was suppressed by norflurazon, which inhibits the carotenoid synthesis. However, treatment of the dark-grown cells with H 2O 2 or methyl viologen did not induce APX.

Pyruvate:NADP + oxidoreductase is stabilized by its cofactor, thiamin pyrophosphate, in mitochondria of Euglena gracilis

Pyruvate:NADP + oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.

Effect of Ni2+ on Early Stages of Chlorophyll Biosynthesis and Pheophytinization in Euglena gracilis

The effect of Ni2+ on the early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis cells was studied. Incubation of the cells with 10–4 M Ni2+ for 7 days resulted in a higher chlorophyll content, enhanced production of 5-aminolevulinic acid (ALA), and in increased activity of 5-aminolevuluinic acid dehydratase (EC 4.2.1.24, ALAD), as compared to the control cells incubated without Ni2+. At a higher concentration (10–3 M), Ni2+ markedly inhibited chlorophyll accumulation and ALAD activity, as compared to the control cells. At this concentration, Ni2+ also inhibited heme biosynthesis and strongly stimulated ALA production. It seems likely that, by affecting heme synthesis, Ni2+ increases the activity of the ALA production system. However, the suppression of subsequent stages of ALA conversion to chlorophyll, in particular ALAD inhibition, ultimately resulted in almost complete inhibition of chlorophyll biosynthesis. In addition to cessation of de novo chlorophyll synthesis in the presence of Ni2+ (10–3 M) in Euglena cells, the existing chlorophyll was converted into pheophytin and almost completely degraded. We suppose that the Ni2+-induced pheophytinization is caused by an acidic shift of intracellular pH related to an impairment of cell membrane permeability by Ni2+ cations.

Nonoptically probing near-field microscopy for the observation of biological living specimens

We present the observation of living specimens with subwavelength resolution by using the nonoptically probing near-field microscopy we have developed recently. In the near-field microscope, the optical field distributions near the specimens are recorded as the surface topography of a photosensitive film, and the topographical distributions are readout with an atomic-force microscopy. Since the near-field microscope does not require the scanning of a probe tip for illumination or detection or scattering of light, it is possible to observe moving biological specimens and fast phenomena. We demonstrate the observation of a moving paramecium and euglena gracilis with subwavelength resolution. The observation of the nucleus inside a euglena cell was also demonstrated.

Detoxification effect of iron-encaging zeolite-processed water in tributyltin-intoxicated Euglena gracilis Z.

In our previous paper, we reported the restoration promoting effects of mineral-encaging zeolite-processed water, especially of a Fe-encaging one, on tributyltin chloride (TBTCl)-intoxicated Euglena gracilis. This present study extends the investigation on the behavior of TBTCl and a xenobiotic enzyme, cytochrome P-450, in Euglena cells incubated with or without Fe-encaging zeolite-processed water (FeZW). Subcellular fractionation of TBTCl-intoxicated Euglena cells, atomic absorption spectrophotometry, and GC analyses showed that TBTCl was rapidly incorporated into the cells to halt cell motility. GC-MS showed that FeZW promoted conversion of TBTCl to dibutyltin (DBT) as the major metabolite in the microsomal fraction of the cells. An in vitro incubation system with heat-treated microsomes did not convert TBTCl to DBT. The contribution of cytochrome P-450 in the microsomal fraction was suggested by an immunochemical method. The results suggest that the improvement of detoxification by FeZW in the TBT-intoxicated Euglena cells should be due to activation of biotransformation system of the Euglena cells by FeZW.

VERTICAL MIGRATION OF EUGLENA SP. ON THE SAND BANKS OF A NORTH CAROLINA PIEDMONT STREAM

On summer days, visible patches of green appeared on the aerially‐exposed sand banks of a small tributary of Mays Lake in Burlington, North Carolina. These green patches became visible approximately 3 hours after sunrise and disappeared 2 hours prior to sunset. Microscopic examination of sediment samples revealed that the appearance of color was due to the upward migration of Euglena sp. that attained densities of 2200 cells per square millimeter around solar noon. Triplicate sediment cores revealed the presence of live Euglena cells as deep as 6 cm below the sediment surface. During the day, over 80% of the population was located between the surface and a depth of 2 mm. During the night, 75% of the population was found between 2 and 6 cm below the surface. Artificially darkening the sediment surface during the day stimulated downward migration of the population. Controlled experiments with neutral density filters indicated that the number of cells on the sediment surface was directly related to incident irradiance. The results of these experiments on Euglena sp. living on the banks of a freshwater stream in central North Carolina will be contrasted with those of previous experiments conducted on Euglena proxima living on the intertidal sand flats of the Newport River estuary in coastal North Carolina.

Orientation of Carotenoid Molecules in the Eyespot of Alga: In Situ Polarized Resonance Raman Spectroscopy

Raman spectra of single cells of Euglena and Chlamydomonas have been examined with 514.5 nm excitation at various points within the cells. At every point, two strong bands, which are assignable to carotenoid, appeared at 1530 and 1159 cm−1. By the use of a Raman mapping system, the Raman intensity at 1530 cm−1 has been plotted against the (x,y) coordinate representing a location within the cell. It has been shown that, for both algae examined, the eyespot has a prominently high carotenoid content, and a small amount of carotenoid is uniformly distributed among the chloroplast. The spatial resolution of the mapping system has been shown to be higher than 1 μm, and the Chlamydomonas eyespot has an elongated shape of 1 μm × 2 μm. By use of a polarizer, the carotenoid chains in the Chlamydomonas eyespot have been found to be aligned along its long axis, which is parallel to the body axis.

Production of pure photosynthetic cell biomass for environmental biosensors

The feasibility of producing pure photosynthetic cell biomass heterotrophically and activating their photosynthetic apparatus through light illumination was investigated using Chlorella sorokiniana and Euglena gracilis. Although the chlorophyll contents of Chlorella and especially Euglena cells decreased during heterotrophic cultivation, photoactivation of the cells resulted in sharp increases in their chlorophyll and some other metabolites contents. Furthermore, the cells swiftly changed from heterotrophic to photoautotrophic metabolism and vice versa, when the culture conditions were cyclically changed from heterotrophic to photoautotrophic. Under this condition, the cells grew continuously and, depending on the light condition during the photoautotrophic phase, there was a stable fluctuation in the intracellular metabolites concentrations. In the case of Chlorella, for example, the protein content of the cells decreased during the heterotrophic phase and then increased during the photoautotrophic phase. The results implied that the cells retained their photosynthetic apparatus during the heterotrophic cultivation and can be easily activated by illuminating the culture. An internally illuminated photobioreactor, which can be used for both heterotrophic cultivation and photoactivation of the cells was therefore constructed. Using this reactor, the cells are first cultivated heterotrophically to a high cell concentration and when the organic carbon source is completely exhausted, the reactor is illuminated for photoactivation of the cells. The photobioreactor has high mass transfer capacity (for efficient heterotrophic culture) and high light supply capacity (for efficient photoactivation). It can be illuminated by both artificial and solar light, can be maintained under strict sterile condition and easy to be scaled up.

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline- N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H + gradient (>60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc 1 complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc 1 complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.

Light-Regulated, Circadian Respiration Activity of Euglena gracilis Mutants that lack Chloroplasts

A circadian photosynthesis rhythm was confirmed in Euglena gracilis wild-type cells by measuring up to eight endogenous oscillations of the concentration of free oxygen in the culture medium under constant light conditions. In colourless Euglena mutants lacking functional chloroplasts, the light dependency and the circadian fluctuations of the free oxygen concentration were inverse to those of wild-type cells. The lowest O 2 concentrations in cultures of the mutant were observed during the daytime, when photosynthesis is maximal in wild-type cells. After synchronisation by 12 h: 12 h light (L) :dark (D) cycles, the mutant cultures showed circadian fluctuations in their oxygen concentration when transferred to constant light. When subjected to a more complex L: D : L: D : L: D regime of 12h:3h:9h: 12h:3h:9h the mutant cells reacted immediately to the light conditions manifested in varying O2 concentrations. Red and blue light cycles of 8 h : 8 h L: D were equally efficient in regulating respiration activity when compared with white light cycles (8h L:8h D) of 16h duration. These results suggest that the demand for energy is under light and circadian control. Whether the energy is supplied by photosynthesis or respiration depends on the Euglena cells tested.

Identification of the catalytic subunit of cAMP-dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z

A gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP-dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4-kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA-selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms.

Effect of Several Solutions Including Mineral-encaging Zeolites on the Restoration of Cell Motility of Tributyltin-intoxicated Euglena gracilis Z.

To examine the detoxification effect of mineral-encaged zeolites on cells impaired by pollutant-intoxication, we used a bioassay system involving Euglena gracilis Z as the model organism and TBTCl as a pollutant. TBTCl exposure causes Euglena cells to quickly change shape from a spherical to spindle form, with the process being reversible by detoxification. Taking advantage of this morphological characteristic, we examined the restoration of motility by water containing zeolites encaging different minerals. TBTCl-intoxicated Euglena cells were incubated in processed water with different types of mineral-encaging zeolites for up to 3 hours. The restoration of motility was evaluated by observing the number of motile cells with a video microscope. Remarkable recovery was observed in the incubation systems with water containing Fe-, Zn-, and Mn-encaging zeolites. However, the effect was suppressed when the water species were treated with the chelator, Chelex-100(®). An equivalent concentration of FeCl3 to that in the Fe-encaging zeolite processed water did not show significant restoration effect.