Chemosensory Responses Toward Oxygen in Euglena gracilis*

SYNOPSIS Euglena gracilis strain Z, at a concentration of 10⁶ cells/ml and in containers of ∽ 0.1-mm thickness, spontaneously forms dynamic ring patterns in the dark. These patterns are modified differentially by illumination with red and with blue light. The red light effect is abolished by treatment with an inhibitor of photosynthesis. Pattern formation is apparently the result of chemophobic responses to oxygen dissolved in the medium. Euglena can respond to both negative and positive concentration gradients, depending upon the absolute magnitude of oxygen concentration. The photo- and chemosensory transduction systems of Euglena interact at a stage which precedes the overt expression of motor responses.     

Light-Induced Body Movement of Euglena gracilis Coupled to Flagellar Photophobic Responses by Mechanical Stimulation*†

SYNOPSIS. In low viscosity media, Euglena gracilis strain Z responds to a sudden change in light intensity by a cessation of forward movement, followed by a reorientation of the locomotor flagellum which results in turning of the cell around the lateral axis (photophobic response). At a viscosity interface between low [～ 1 cP (centipoise)] and high (4000 cP) media, the cells exhibit avoidance responses or become immobilized in the higher viscosity medium. Upon changing the light intensity, free swimming cells have photophobic responses, while immobilized ones undergo body contractions. For cells immersed in media of varying viscosity, the delay between light stimulation and body contraction (transduction time) is shortest at high viscosities. From 500 to 2000 cP, where the cells are capable of both movement and light-induced body contractions, there is a logarithmic dependence of the transduction time on the viscosity. The transduction time does not vary appreciably with the intensity of the primary light stimulus within a range of 0.14-1.13 kW/m².     

High Speed Cinemicrography of the Direct Photophobic Response of Euglena and the Mechanism of Negative Phototaxis*

SYNOPSIS. The shock reaction of Euglena gracilis strain Z to a sudden increase in light intensity (the “direct photophobic response”) was examined by high speed cinemicrography. The response is expressed as a turning reaction toward the dorsal side of the cell, after a transduction time of 0.1–0.5 sec after the onset of stimulation. Transduction times, turning rates, and flagellar beat frequencies were measured by analyzing the filmed sequences. The experimental data are consistent with a mechanism of directional homeostasis in negative phototaxis that is based upon shading of the photoreceptor by the cell's posterior end.     

A Photorepressible Isozyme o Malic Enzyme in Euglena gracilis Strain Z*†

SYNOPSIS. Isozymes of malic enzyme in Euglena gracilis strain Z were analyzed by starch-gel electrophoresis. Wild-type and heat-bleached strains were cultured in the light and the dark in the presence of various carbon sources. An isozyme detectable in heterotrophic cultures was repressed by photosynthesis. A model is proposed to explain photorepression of this isozyme.     

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.     

Cyclic Changes in Chloroplast Structure in Synchronized Euglena gracilis*

SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.     

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.     

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.     

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.     

Circadian Rhythm Changes in Autotrophic Euglena Induced by Organic Carbon Sources*

SYNOPSIS. Acetate added to autotrophic Euglena cultures changed the period length of the circadian rhythm of phototaxis. Phase shifts were induced by acetate pulses. Since transition from one metabolic state to another (autotrophic/mixotrophic) caused a phase shift or a period change, such effects possibly result from switching metabolic pathways. As suggested (Brinkmann, K., 1966. Planta 70 , 344–89), differences in the temperature responses of the rhythm in mixotrophic and autotrophic cells might also be caused by participation of different metabolic pathways with different Q¹⁰ values, e.g. dark reactions vs photochemical reactions. However the Q¹⁰ of a given dark reaction, e.g. protein synthesis, can differ in the 2 states. Therefore temperature experiments alone do not suffice for deciding whether the pathways include photochemical reactions, dark reactions, or both.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

Hydrolytic Enzymes of Euglena gracilis: Characterization and Activity as a Function of Culture Age and Carbon Deprivation*†

SYNOPSIS. Optimal assay conditions are described for 8 hydrolases of Euglena gracilis var. bacillaris, SM-L1 (streptomycinbleached) strain, 7 of which have an acid pH-optimum. Acid phosphatase, β-galactosidase, β-glucosidase, β-fucosidase, cathepsin D, RNase, DNase, and an esterase are active in cell homogenates. Amylase has very low activity, and β-glucuronidase, arylsulfatase, β, N-acetyl-glucosaminidase, α-fucosidase, and α- and β-mannosidase are inactive. Hydrolase activity increases as a culture proceeds from the midexponential to the late stationary-phase of growth, being most pronounced in the case of β-glucosidase. In cultures deprived of a utilizable carbon source, the specific activities of the hydrolases (per mg total protein or dry weight) increase. When expressed on a per cell basis, however, the activities of DNase decrease while those of β-galactosidase, cathepsin D, and RNase increase. The hydrolases appear to be involved in the adaptation of Euglena to the metabolic demands imposed by different conditions of growth.     

Aldolase Levels in Wild-Type and Mutant Euglena gracilis

SYNOPSIS. Wild-type Euglena gracilis var. baciliaris , strains B and Z, synthesize 2 distinct fructose-1,6-diphosphate aldolases. Amounts of the 2 activities depend upon conditions of growth. The class I enzyme, with activity similar to that found in various photosynthetic tissues, is formed during regreening of dark-grown cultures incubated in the light. Very low activity of the class I enzyme is also found in mutant strains W₃BUL and W₈BHL, both of which apparently lack plastid DNA. The significance of these findings is discussed.     

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.     

Phénomènes Périodiques, Métaboliques et Structuraux Chez un Protiste, Euglena gracilis

RESUME. Des divisions synchrones chez Euglena gracilis Z peuvent ětre obtenues par différentes méthodes. Lorsque les cellules sont cultivées sur milieu contenant du lactate comme seule source de carbone, des divisions synchrones sont observables, indépen-damment des conditions d'éclairement. Toutefois, il existe une relation entre la phase des divisions cellulaires et les périodes lumière-obscurité appliquée à la culture. Pendant le cycle cellulaire, nous montrons que les synthèses des macromolécules sont discontinues: c'est le cas pour les ADN nucléaire et mitochondrial, les ARN ribosomaux et non-ribosomaux, ainsi que pour certaines protéines (cytochrome c 558). Des variations cycliques touchant la morphologie des mitochondries et des chloroplastes sont observées. Au cours du cycle cellulaire, les processus métaboliques séquentiels accompagnent les modifications de structure des organites. C'est ainsi qu'en début du cycle, au commencement de la phase G₁, sont synthétisés les ribosomes cytoplasmiques et qu'ensuite, chez les euglènes vertes, les ARN non-ribosomaux sont formés. Ces synthèses d'ARN précèdent l'accroissement du chondriome et du plastidome dans la cellule. En milieu de phase G₁, une nouvelle synthèse d'ARN non-ribosomal commence et est observée avant la synthèse des ADN nucléaire et mitochondrial. En fin de phase G₁, démarre la division des organites à partir du chondriome et du plastidome en réseau. SYNOPSIS. Synchronous divisions of Euglena gracilis strain Z can be obtained by various methods. When the cells are cultivated in a medium containing lactate as the sole carbon source, synchronous divisions are observed, independent of the conditions of illumination. Nevertheless, there exists a relationship between the phase of cell division and the periods of light and darkness applied to the culture. During the cell cycle, the synthesis of macromolecules is discontinuous—this is true of nuclear and mitochondrial DNA, ribosomal and nonribosomal RNA, and certain proteins (cytochrome c 558). Cyclic variations in the structure of mitochondria and chloroplasts are also observed. In the course of the cell cycle, sequential metabolic processes accompany structural modifications of the organelles. Also, at the beginning of the cycle, at the start of phase G₁, the cytoplasmic ribosomes are synthesized, and then, in green euglenids, nonribosomal RNAs are formed. These syntheses of RNA precede enlargement of the chondriome and plastids. In mid-G₁ phase, a new synthesis of RNA begins, which precedes synthesis of nuclear and mitochondrial DNA. At the end of G₁ phase, division of organelles starts, beginning with the chondriome and plastids, arranged in a network.     

The Molecular Biology of Euglena gracilis. VIII. Glycitols and Monosaccharides

Mannitol is identified as the major component of the small molecule pool of Euglena gracilis strain Z. Smaller amounts of glycerol also are found. Results of tracer studies and enzymic measurements indicate that both major mannitol pathways found in bacteria are present in Euglena.     

Phylogenetic Origin of the Chloroplast*†

SYNOPSIS. The 16S ribosomal RNA of the chloroplast of Euglena gracilis strain Z has been characterized in terms of its 2-dimensional electrophoretic “fingerprint” (T1 ribonuclease). Over 100 spots were resolved on the “fingerprint” and each spot was characterized as to which RNA oligonucleotide fragment(s) it contained. When compared to similar analyses of prokaryotic 16S rRNAs and eukaryotic cytoplasmic 18S rRNAs, the chloroplast 16S rRNA was a typically prokaryotic RNA, but bore little if any relationship to eukaryotic 18S rRNAs. Therefore, the cistrons for chloroplast 16S rRNA are related to the equivalent prokaryotic cistrons, but, apparently, are not related to the equivalent eukaryotic cistrons. Among the organisms available for comparison, the Euglena chloroplast 16S rRNA appears most closely related to the 16S rRNA of the eukaryote, Porphyridium cruentum (a red alga), and at least distantly related to the 16S rRNAs of the blue-green algae and perhaps also to the bacilli.     

ATPase Activity in Flagella from Euglena gracilis. Localization of the Enzyme and Effects of Detergents*

SYNOPSIS. The biochemical effects of some detergents on the ATPase activity of isolated flagella from Euglena gracilis are related to morphologic obliterations induced by those detergents. Enzymic activity can be localized by electron microscopy along the microtubules and also on the paraflagellar rod. The nonionic detergent digitonin solubilizes the enzyme linked to dyneinic arms, whereas the activity linked to residual structures appears enhanced. These results support the hypothesis that the paraflagellar rod may be a structure actively related to the motility of this type of flagellum.     

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.