Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

Influence of Culture pH on Chloroplast Structure in Euglena gracilis

Chloroplasts of Euglena gracilis grown with phototrophic nutrition at pH 3.0 were compact, while those in cells grown at pH 8.1 were swollen with widely separated lamellae.     

The natural herbicide, cyanobacterin, specifically disrupts thylakoid membrane structure in Euglena gracilis strain Z

Abstract Cyanobacterin is a natural product produced by the cyanobacterium (blue-green alga), Scytonema hofmanni . The compound has been chemically characterized and shown to inhibit electron transprot in photosystem II. Although the herbicide is lethal to photoautotrophs, photoheterotrophically-grown organisms such as Euglena gracilis can survive and grown in saturating concentrations of cyanobacterin. Electron micrographs of treated E. gracilis cells show extensive damage to the thylakoid membranes of the chloroplasts, similar to the effects observed with 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU). Unlike the synthetic herbicide, cyanobacterin specifically disrupts thylakoid membranes and does not affect other cellular membranes or heterotrophic growth.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

Some Biochemical Properties of Mitochondria Isolated from Euglena gracilis*

SYNOPSIS. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only ～ 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg²⁺-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and and UTP; with Ca²⁺ in place of Mg²⁺ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was ～ 7 regardless of the uncoupler used, and ～ 8 without the uncouplers. The few differences observed between mitochondria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

Stimulation of Plastidogenesis Induced by 5-Azacytidine in Euglena gracilis Klebs

When etiolated Euglena gracilis was treated with 10 mM 5-azacytidine (5-azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub-population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LB_S). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple-layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5-azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LB_S opens the possibility of studying early aspects of plastid development in Euglena.     

Subcellular Localization of the GABA-Shunt Enzymes in Euglena gracilis Strain Z

SYNOPSIS. Glutamate decarboxylase, γ-aminobutyrate-α-ketoglutarate aminotransferase and NAD-linked and NADP-linked succinic semialdehyde dehydrogenase, all constituting the GABA (γ-aminobutyrate)-shunt pathway of glutamate metabolism are localized in the mitochondrial matrix in a streptomycin-bleached mutant of Euglena gracilis strain Z. Glutamate dehydrogenase, requiring NADP as the cofactor, was distributed in the cytoplasm. An improved version of the controlled digestion method for preparing Euglena mitochondria, which involves use of trypsin and a trypsin inhibitor and removal of broken cells before mechanical disruption of cells, is also described.     

Isolation of the photoreceptor (paraflagellar body) of the phototactic flagellate Euglena gracilis

We have described a procedure for isolating photoreceptors (paraflagellar bodies) from the unicellular phototactic alga Euglena gracilis. This procedure elicited the evagination of the reservoir membrane, and the detachment of the flagellar apparatus due to the very high concentration of CaCl₂ in the isolation solution that caused a frenetical acceleration of the flagellar beating.     

The Molecular Biology of Euglena gracilis IX. Amino Acid Pool Composition

The amino acid composition of the acid soluble fraction of Euglena gracilis was determined from cells grown in 4 different culture media. Glutamic acid is the major free amino acid. Hydrolysis of this fraction increases the amount of free amino groups, the major amino acids found are then glutamic acid, aspartic acid, glycine and arginine. The pattern of amino acid distribution is similar in all 4 culture media. L-arginyl-L-glutamine was isolated and identified in extracts from all 4 culture conditions. It was shown to be a metabolic intermediate by radioactivity chase experiments.     

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

Multiple effects of salinity on photosynthesis of the protist Euglena gracilis

The effect of NaCl in the culture medium on growth, photosynthesis and cell content of chlorophyll, K⁺, Na⁺, Ca²⁺ and Mg²⁺ in Euglena gracilis was studied. O₂ production, quantum yield of photosystem II (PSII), the non-photochemical quenching of chlorophyll fluorescence (q_N) and the chlorophyll alb ratio all diminished by 0.2 M NaCl. Respiration and chlorophyll a and b increased, whereas the photochemical quenching (q_p) of chlorophyll fluorescence was not affected by 0.2 M NaCl. Salt stress also induced an increase in cell volume and in K⁺ and Na⁺ concentrations, but decreased the concentrations of Ca²⁺ and Mg²⁺. Except for a protective effect on O₂ production, additional Ca²⁺ in the culture medium did not attenuate the salt effect on the parameters measured. The addition of HCO₃^? restored the PSII quantum yield of O₂ production in cells grown in high salt. Salt stress promoted a decrease in the apparent rate of quinone A (Q_A) reduction and an apparent obstruction of Q_B reduction, which were not prevented by excess HCO₃^?; the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not increase chlorophyll fluorescence in salt-grown cells. These results indicate that photosynthesis in Euglena grown under salt stress exhibits: (1) diminution of the HCO₃^? dependent water-splitting activity of PSII; (2) inhibition of the electron transfer at the quinone pool level; (3) probable increase in thylakoid stacking (as indicated by the effect on the chlorophyll alb ratio); and (4) dissipation of the H⁺ gradient across the thylakoid membranes (as indicated by the decrease of q_N).     

Euglena gracilis as source of the antioxidant vitamin E. Effects of culture conditions in the wild strain and in the natural mutant WZSL

The photosynthetic wild type and the spontaneous non-photosynthetic WZSL mutant of the unicellular flagellate Euglena gracilis were grown to investigate the influence of photoheterotrophic and heterotrophic conditions on α-tocopherol (vitamin E) content. HPLC analysis demonstrated a marked enhancement (almost 100%) of tocopherol content in the light in both strains, independent of the presence of chloroplasts. These findings indicate that the formation of vitamin E occurs inside both the mitochondrial and chloroplastic compartment, and that the correlation between light and vitamin E production is not linked to the existence of chlorophyll. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Molecular Biology of Euglena gracilis. X. Amino Acid Composition of Protein

SYNOPSIS. Cells of Euglena gracilis strain Z were extracted with trichloroacetic acid. Samples of gross cellular protein were hydrolyzed by a variety of reagents. Amino acids released by these procedures were analyzed and the overall composition of cell protein was quantitatively determined.     

Protein Synthesis in Euglena gracilis is Light-and Temperature-Dependent,Oscillating in a Circadian,Temperature-Compensated Manner

Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of ³⁵S-methionine- as well as ³⁵S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C.     

Mono-ADP-Ribosylation of Arginine Residue of Euglena gracilis Z in Synchronous Culture

ABSTRACT. In Euglena gracilis Z, a considerably high activity of mono-ADP-ribosyltransferase occurred and change of it was accompanied by a cell cycle induced by a light-dark cycle. The enzyme activity was strongly inhibited by L-arginine and supported in the presence of poly-L-arginine as a substrate, indicating that ADP-ribosylated amino acid is an arginine residue. Arginine: mono-ADP-ribosyltransferase activity was found in the chloroplasts, mitochondria, microsomes and cytosol as judged from marker enzyme activities and the activity in each organelle fluctuated with the cell cycle.     

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)_4–6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)₂ and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)₂ or GlcNAc, which are easily taken up by the cells.     

In vivo and in vitro methylation of the elongation factor EF-Tu from Euglena gracilis chloroplast

Based on amino acid sequence similarities between the methylated elongation factor EF-Tu from Escherichia coli and the EF-Tu from Euglena gracilis chloroplast, we predicted that the latter could also be methylated in the presence of an appropriate methyltransferase. We found that, as reported for the eubacterial homologous protein, the organellar factor could be methylated in vivo and in vitro to yield monomethyllysine.     

The Relationship of Fixed Carbon and Nitrogen Sources to the Greening Process in Euglena gracilis strain Z*

SYNOPSIS The pattern of chloroplast development was followed in Euglena gracilis strain Z greening in media with a variety of fixed carbon and nitrogen sources. The greening pattern of cells grown in inorganic medium with added ethanol or glucose involves an inhibition of chloroplast development when compared to that of cells grown in inorganic medium alone. Several nitrogen sources were tested to ascertain their effectiveness in relieving the inhibition of chloroplast development by glucose. Of those, only 0.05% (w/v) (NH₄)₂ SO₄ accelerated the recovery from the inhibition after most of the glucose had been removed from the medium by the cells. The other nitrogen sources tested were not effective. An inhibition of chloroplast development, similar to that observed in cells greening in the presence of glucose, was seen in cells greening in an ethanol-containing medium. These cells, however, had a different response upon the addition of 0.05% (NH₄)₂ SO₄. They appeared to recover from the inhibition of chloroplast development, even before the ethanol was removed from the medium by the cells. A slight enhancement of chloroplast development was noted in cells greening in an inorganic medium with glycine or serine. Other amino acids tested had little or no effect.     

Regulation by Ammonium of Variation in Heterotrophic CO2 Fixation by Euglena gracilis During Growth Cycles*

SYNOPSIS Heterotrophic (dark) CO₂ fixation by Euglena gracilis strain Z varies with phase of batch culture growth and mode of nutrition. Increases in the fixation during growth cycles correlate closely with the depletion of exogenous NH₄* from the medium during growth. It is demonstrated that exogenous NH₄⁺ regulates a component of heterotrophic CO₂ fixation and that another component is independent of NH₄⁺. This is true for cells grown heterotrophically (glucose, dark), autotrophically (CO₂, light) and for a permanently bleached strain (E. gracilis SB3). Some kinetics of the NH₄⁺ regulation are presented.