Pellicle structure in Euglena

The pellicle of Euglena has been investigated by anoptral and phase contrast light microscopy, and by electron microscopy of osmium-fixed, Epon-embedded, lead-stained sections and of carbon/platinum replicas.

Observations on living cells show that the pellicular striatiors of Euglena spirogyra trace a left-handed (S) helix in a majority of cells, and a right-handed (Z) helix in only 5 to 30% of the cells in any one culture. All cells of E. spirogyra var. fusca have a left-handed (S) helix. Ornamentation on the pellicle takes the form of rows of knobs in various patterns. The living pellicle can be dissociated into long flat strips.

Electron microscopy shows that each pellicular strip has an elaborate cross-sectional shape, features of which are accessory teeth and ribs and a continuous ridge which articulates in a groove running along the edge of the next strip. The strips can move against one another, presumably by the ridges sliding in the grooves, and it is suggested that the joints might be lubricated by mucilage supplied from the helically disposed muciferous bodies. One single and one pair of fibrils or tubes, 200–250 Å in diameter, are regularly arranged parallel to each pellicular strip. A continuous tripartite plasmalemma, 80–100 Å thick, lies externally to the strips; external to this membrane lie the pellicular knobs. Each cell has from 35 to 45 pellicular strips, reducing to a few at the posterior end of the cell by successive fusions. Similar fusions occur at the anterior end of the cell, mainly within the canal.

These observations are compared with those made on the euglenoid pellicle by previous authors, and the following problems are discussed: direction of helix; the nature and cause of ornamentation; euglenoid movement with reference to fibrils, cytoplasmic flow, pellicle flexibility and the proteinaceous nature of the pellicle; helical and bilateral symmetry in the cell; and cet growth and division.     

Effect of Nitrofurans on the Chloroplast System of Euglena gracilis

SYNOPSIS. Twelve derivatives of 5-nitrofuran were tested on Euglena grarzlis . All rendered the organism permanently apoplastidic and, at somewhat higher concentrations, killed. The furan analogues of 3 of these compounds had no effect on the chloroplast system and were less toxic than the nitrcfurans. Low concentrations of nitrofurantoin and nitroiuraldehyde inhibited formation of chlorophyll when etiolated cells irere illuminated.
Exposure of euglena to low concentrations of these agents for about 2 generation times. followed by plating on drug-free mediurn. resulted in a high proportion of bleached colonies. It is theretore concluded that the nitrofurans induce apotplastidy by causing permanent damage to the chloroplast system rather than by inhibiting its replication temporarily. Since one of the nitrofurans which was found to bleach euglena, NFT–3-amino-6[-2-(5-nitro-2-furyl) vinyl]-1,2,4-triazine–is known to cause specific inhibition of DNA synthesis in bacteria, nitrofurans may perhaps bleach euglena through selective damage to chloroplast-DNA or to the DNA-synthesizing system of the chloroplast.     

Effect of Light on Glucose Utilization by Euglena gracilis

The effect of light on glucose consumption by wild-type Euglena gracilis Z. and mutant cells has been studied. When dark- or light-grown wild-type cells are transferred from a medium containing sodium butyrate as the only carbon source to a glucose-containing medium, glucose consumption is blocked for 6 to 7 days when cultures are incubated under a light intensity of at least 600 lux. During this time cells multiply at the same rate as controls kept on media devoid of any utilizable organic carbon source. This light-induced inhibition of glucose consumption and of growth on glucose-containing medium is not related to photosynthesis since: (a) glucose consumption is inhibited by light intensities much lower than those required for high phototrophic growth; (b) the inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea does not overcome the inhibition of glucose consumption; and (c) nonphototrophic-growing mutants also show light-induced inhibition of glucose consumption and of growth on glucose-containing medium. This inhibition of growth by light might be explained by modification in the permeability of the cellular membrane.     

The photoreceptor protein of Euglena gracilis

We isolated the photoactive protein Erh, isolated from the photoreceptor of the unicellular photosynthetic flagellate Euglena gracilis. It is a 27 kDa protein with a photocycle resembling that of sensory rhodopsin, but with at least one stable intermediate. We recorded the absorption spectrum of the parent form of this protein both under native form and in the presence of hydroxylamine and sodium borohydride, and the fluorescence spectra of both the parent and intermediate forms. We suggest that Erh is a rhodopsin-like protein and propose a simple photocycle. This protein shows optical bistability, without thermal deactivation.     

The Warburg Effect in a Chloroplast-Free Preparation from Euglena gracilis

A supernatant fraction, free of plastids, was prepared by centrifugation from Euglena gracilis and used to ascertain whether or not the inhibition of carbon dioxide fixation by oxygen, known as the Warburg effect, is entirely independent of the light-driven phase of photosynthesis. This fraction exhibited in the dark the main features of the Warburg effect; namely, an inverse relationship between the degree of inhibition by oxygen and bicarbonate concentration, reversibility of the inhibition when the oxygen partial pressure is lowered and an increase in the proportion of 2-carbon compounds. It is proposed, therefore, that the inhibition by oxygen is manifest in the photosynthetic carbon reduction cycle and is independent of photosynthetic electron transport and phosphorylation.     

The alternative oxidase from Euglena gracilis

We still have a rudimentary understanding about the mechanism by which plant roots may stimulate soil microbial interactions. A biochemical model involving plant-derived biochemical fractions, such as exudates, has been used to explain this "rhizosphere effect" on bacteria. However, the variable response of other soil microbial groups, such as protozoa, to the rhizosphere suggests that other factors could be involved in shaping their communities. Thus, two experiments were designed to (a) obtain a better understanding of the mechanism by which ciliate species richness and abundance differ among plant species and (b) to determine whether this mechanism is maintained via stimulatory and/or inhibiting factors associated with particular plant species. Bacterial and chemical slurries were reciprocally exchanged between two plant species known to differ in terms of ciliate species richness and abundance (i.e., Canella winterana and plantation Tectona grandis ). The ANOVA showed that the bacteria plus nutrients, and the nutrients-only treatment have no significant effect on the overall ciliate species richness and abundance when compared to the control treatment. However, the use of only colpodean species to increase the taxonomic resolution of treatment effects showed that bacterial slurries have a significant effect on colpodean ciliate species richness. These results suggest that for particular rhizosphere ciliates, biological properties, such as bacterial diversity or abundance, may have a strong influence on their diversity and possibly abundance. These results are consistent with a model of soil bacteria-mediated mutualism between plants and protozoa.     

Effect of Ultraviolet Irradiation on DNA Metabolism of Euglena gracilis

SYNOPSIS. (P³²O₄) is rapidly incorporated into DNA of resting cells of Euglena. This incorporation is strongly inhibited by bleaching doses of ultraviolet light. Photoreactivation of the bleaching effect does not immediately restore the rate of P³² incorporation into DNA. It is indicated that this metabolically active DNA is not directly associated with control over the development of the plastids.     

Growth of Euglena gracilis on whey

Summary To extend the use of industrial wastes, we have studied the growth of Euglena cells on demineralized whey powder, an industrial dairy waste from cheese making. The demineralized whey powder was solubilized (15 g/l) in 0.04 N HCl and autoclaved for two hours at 120°C. The solution was then brought to pH 3.5 with NH₄OH and tested for its ability to support Euglena growth. In the dark, cell densities of 4.5 to 5.5×10⁶ cells/ml were obtained when vitamin B₁₂, thiamine and minerals were added to the hydrolyzed whey solution. Although growth of Euglena is possible on whey, the industrial application may be limited due to the need to hydrolyze the whey and to the low utilization of carbon (20%) as the glucose, but not the galactose, released during hydrolysis is used.     

Heteroxanthin in Euglena gracilis

Summary 1. From a large scale preparation of Euglena gracilis, strain Z, besides the acetylenic pigments diatoxanthin and diadinoxanthin and the allene neoxanthin, an additional acetylenic xanthophyll has been isolated. 2. Mass and IR spectra and chemical reactions showed typical patterns of heteroxanthin from Vaucheria. 3. The pigment was transformed into diadinochrome-isomers with acidified acetone. 4. A partial synthesis of heteroxanthin from diadinoxanthin by LiAlH₄-reduction is described, confirming the structure proposed by Strain. 5. The identity of heteroxanthin with the trollein—like pigment described for Euglena is discussed.     

The effect of rapamycin on biodiesel-producing protist Euglena gracilis

Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.     

Effect of Cobalamin Deficiency on the Biosynthesis of Phosphatidylcholine in Euglena gracilis

Euglena gracilis requires cobalamin (Cbl) as an essential growth factor. Phosphatidylcholine (PC) synthesis was greatly reduced by Cbl deficiency. Rapid cell division occurred after Cbl was replenished, and PC was actively synthesized during the cell divisions. When the deficient cells were given methionine (a precursor for the choline moiety), active synthesis of PC occurred even without the Cbl supplement, although cell division was not induced. As methionine synthase in Euglena requires methylcobalamin as a coenzyme, decrease in methionine synthesis may account for reduced PC synthesis under Cbl-deficient conditions. Phosphatidyleth-anolamine and phosphatidylserine synthesis were also suppressed, commensurate with decrease of PC synthesis, under Cbl deficiency, even though Cbl is not thought to participate in their synthesis. In contrast, a lot of triglyceride and wax ester accumulated in Cbl-deficient cells. Moreover, Cbl depletion altered fatty acid composition, notably due to increased proportion of odd-numbered fatty acids     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyltransferRNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 g/ml), all three log phase enzymes showed an increase in activity of 40–50%. At slightly higher concentrations (2.5 g/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5g/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5–20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5g/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10g/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.     

Antimutagenicity in Euglena gracilis

The genotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, α-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis. The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100 and TA102, were used. The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102.