Inhibition by Chloramphenicol of Chlorophyll and Protein Synthesis and Growth in Euglena gracilis

SYNOPSIS. Chloramphenicol inhibits growth of Euglena gracilis. It also interferes in the greening process by inhibiting protein synthesis (measured by leucine uptake), but RNA synthesis is essentially unafiected. The antibiotic acts on all cell fractions, but prefermtially inhibits the synthesis of plastid protein. Inhibition of chlorophyll synthesis is transitory and does not affect the self-reproduction system for chloroplast formation.     

Growth Inhibition by Phenylalanine in Euglena gracilis

The hydrolysis and esterification by a thermostable lipase from Humicola lanuginosa No. 3 were investigated. Both reactions occurred readily at temperatures between 45~50°C. Esterification by the enzyme with glycerol was observed to be specific towards fatty acids with carbon numbers of C12~C18. Laurie acid esters with different alcohols such as primary alcohols, terpene alcohols, eie., were also synthesized readily. Esterification by the enzyme was adversely affected by the water content (optimum, ca. 7%), however, the hydrolysis rate increased rapidly with increasing water content (optimum, az. 60%). The enzyme showed increased activity in organic solvent-aqueous reaction systems. Nevertheless, hydrolysis in complete organic phase reactions was found not to be feasible. Hydrolysis at a higher temperature (50 or 55°C) in a solvent free phase was almost the same as that in organic solvent-aqueous phase reactions. The components of glycerides varied considerably during hydrolysis, whereby esterification resulted in a higher quantity of mono- and diglycerides (about 40%), compared to in the case of hydrolysis, for which the value was about 10~20%.     

Chlorophyll Formation in Euglena gracilis var. bacillaris: Effect of Chelating Agents

SYNOPSIS. 8-Hydroxyquinoline (8-HQ) and cysteine markedly inhibit the synthesis of chlorophylls in non-proliferating etiolated Euglena gracilis var. bacillaris on illumination. This is thought to be due, at least to some extent, to the binding of Mg²⁺ ions in the non-proliferation medium, but largely as a consequence of intracellular chelating action. The effect of 8-HQ could be reversed by the presence of metal ions higher in the Mellor-Malley series than Mg²⁺, e.g., Zn²⁺ and Co²⁺. Sodium diethyldithiocarbamate (Na-DDG) and ethylenediaminetetraacetic acid (EDTA) have no effect on chlorophyll synthesis in non-proliferation conditions. Growth in the light is more affected than growth in the dark by all these chelating agents, including Na-DDG and EDTA. This is due to the differential requirement of Mg²⁺ under these 2 growth conditions.     

Chlorophyll Formation in Euglena gracilis var. bacillaris: Effect of Enzyme Inhibitors

SYNOPSIS. The synthesis of chlorophylls in non-proliferating dark-grown etiolated cells of Euglena gracilis var. bacillaris is markedly inhibited by the enzyme inhibitors dinitrophenol (DNP), p -chloromercuribenzoate (pCMB) and sodium fluoride. The pCMB inhibition of greening is reversed to a great extent by glutathione but not by cysteine. The inhibitory effect of fluoride is reversed by both succinate as well as Mg⁺⁺ during illumination. Fluoride also inhibits the photosynthetic growth of the euglenas; this inhibition is competitively reversed by succinate.     

Chlorophyll fluorescence quenching in the alga Euglena gracilis

When far red light preincubated cells of Euglena gracilis are transferred to dark or light, chlorophyll fluorescence (F₀ and F_m) decreases. Non-photochemical quenching in the dark is suggested to be induced partly by chlororespiration and partly by changes in the distribution of excitation energy between the photosystems. Depending on the light intensities it was possible to resolve the non-photochemical quenching into at least three different components. The slowest relaxation phase of non-photochemical quenching occurred only after exposure to high light and was assigned to photoinhibition. The other two components were an energy-dependent quenching (qE), and the one which we attribute to a spill over mechanism. We suggest that both photosystems use a common antenna system consisting of LHC I and LHC II proteins. In contrast to higher plants, qE in Euglena gracilis is independent of the xanthophyll cycle and an aggregation of LHC II. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Phosphorus and the Growth of Euglena gracilis

SYNOPSIS. Growth of streptomycin-bleached Euglena gracilis depends in part on the availability of phosphorus. Maximum cell density on a defined medium is reached at a phosphorus (supplied as inorganic phosphate) concentration of 4–5 μ/ml. At lower concentrations, the cells apparently deplete the medium of phosphorus. Inorganic phosphorus at > 1 mg/ml inhibits growth in terms of cell density per ml and generation time. Phosphorus-limited cells survive for at least 6–7 days and are able to undergo mitosis following a lag period when returned to phosphorus-containing medium. The majority of inorganic ³²P incorporated by these Euglena ends up in the hot trichloroacetic acid soluble fraction and in the ethanol-ether soluble fraction.     

Relative Requirements for Magnesium of Protein and Chlorophyll Synthesis in Euglena gracilis

The relationships among Mg, growth, chlorophyll synthesis, and cytoplasmic polysome content were studied in Euglena gracilis grown in different levels of the metal. At all levels of magnesium from 20 to 1,600 μmolar, both protein and chlorophyll are formed with exponential kinetics. The apparent rates of synthesis and final yields of both components are greater at higher levels of Mg, but the rate of chlorophyll synthesis always exceeds the rate of protein formation; i.e. the most severely deficient cells contain proportionally more chlorophyll than the sufficient cells. Cytoplasmic polysomes isolated from Mg-deficient Euglena are indistinguishable from those isolated from control cells. We conclude that decreased rates of protein synthesis occur prior to and possibly are causal to decreased rates of chlorophyll synthesis, but that the mechanism of this inhibition remains unclear.     

A possible ribosomal-directed regulatory system in Euglena gracilis. Chlorophyll synthesis

Cycloheximide at concentrations of 0.1-100mum stimulated chlorophyll synthesis when dark-grown cells of Euglena were illuminated. Chloramphenicol (1-4mm) inhibited chlorophyll synthesis. The effect of cycloheximide on the incorporation of [(14)C]leucine into material insoluble in trichloroacetic acid, and its failure to affect the incorporation of [(32)P]orthophosphate into such material in short incubations, are interpreted as evidence that cycloheximide specifically inhibits protein synthesis by 80S ribosomes. Since the inhibitory effect of chloramphenicol on chlorophyll synthesis is counteracted by the presence of cycloheximide, it is suggested that chlorophyll synthesis is subject to control by a cytoplasmic repressor synthesized on 80S ribosomes, and to a de-repressor synthesized on 70S ribosomes.     

Light-induced Enzyme Formation in a Chlorophyll-less Mutant of Euglena gracilis

A mutant strain, Y₉, of Euglena gracilis strain Z that is unable to produce protochlorophyll or chlorophyll has been isolated following treatment of wild type cells with nalidixic acid. Dark-grown cells of the mutant contain proplastids that show only limited ultrastructural development when placed in the light. Treatment of Y₉ cells with ultraviolet light brings about permanent cell bleaching with a target number similar to wild type Euglena, and with a slightly greater sensitivity to ultraviolet. Three enzymes of the reductive pentose phosphate cycle, fructose-1,6-diphosphate aldolase (class I), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, are detectable in dark-grown Y₉ cells at the low concentrations characteristic of dark-grown wild type cells, and increase substantially when these cells are exposed to light. The activity of ribulose-1,5-diphosphate carboxylase increases in the light to a lesser extent. Cytochrome 552, a carrier in the photosynthetic electron transport chain, is not present in light-grown cells of Y₉. The significance of this mutant for an understanding of the role of light in Euglena chloroplast development is discussed.     

Study on the C5-Pathway of Chlorophyll Biosynthesis in Euglena gracilis Klebs

By feeding (l-13C)-glutamic acid to Euglena gracilis, a green alga, the 13C-NMR spectra, of chlorophyll showed that the entire carbon skeleton of glutamic acid was incorporated into chlorophyll. The existence of cspathway in chlorophyll biosynthesis was proved. In cell-free test, porphobilinogen (PBG) was formed from (5-13C)-δ-aminolaevulinic acid(ALA), but no evidence for (2-13C)-glycine incorporation was observed. It means that the Shemin pathway was effectivelessness, at least, in E. gracilis.     

Growth and cell volume of Euglena gracilis in different media.

It is necessary to propagate Euglena gracilis cells for several days after transfer from one medium to another to establish the steady state of balanced growth. Steady-state growth was established in minimal and in complex medium. Specific growth rates and cell volume distributions were computed for each culture medium. Mean cell volume of E. gracilis is not uniquely correlated with the specific growth rate.     

Growth and cell volume of Euglena gracilis in different media.

It is necessary to propagate Euglena gracilis cells for several days after transfer from one medium to another to establish the steady state of balanced growth. Steady-state growth was established in minimal and in complex medium. Specific growth rates and cell volume distributions were computed for each culture medium. Mean cell volume of E. gracilis is not uniquely correlated with the specific growth rate.     

Growth in Volume of Euglena gracilis During the Division Cycle

The distribution of volumes of Euglena gracilis cells was measured conductimetrically. The volume spectrum of cultures in balanced growth was analyzed by the method of Collins and Richmond. The kinetics of volume increase of Euglena is neither linear nor exponential; the growth rate of small and large cells is low, but intermediate size cells show the largest growth rate.     

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.     

Degradation of Paramylon by Euglena gracilis

SYNOPSIS. Protozoa of the order Euglenida contain a polysaccharide storage product, paramylon, composed of 1, 3-linked glucose molecules arranged into an extremely resistant granule. An enzyme was purified from the soluble phase of Euglena gracilis which would degrade this polysaccharide to single glucose residues, providing the integrity of the paramylon granule was 1st disrupted by dilute base. This enzyme, a β-1, 3 glucanase, had optimal activity at pH 5.0 and 60 C and bound tightly to base-disrupted paramylon substrate tho not to the intact granules. The specific activity of the enzyme was doubled when cell cultures reached stationary phase, the phase where net carbohydrate utilization began. An ATP-dependent hexokinase reaction was also present in Euglena homogenate. No phosphorylase activity has been found in Euglena. It is suggested, therefore, that Euglena do utilize their paramylon as a carbohydrate reserve and the mechanism of this utilization is by exo-hydrolytic cleavage to free glucose followed by phosphorylation and glycolysis.     

On Hemoglobin Inhibition of the Growth of Euglena gracilis

SYNOPSIS. The inhibitory principle in whole blood for strains of Euglena gracilis has been characterized and identified as hemoglobin. Hemoglobins from different sources were tested for their inhibitory activity and gave similar results. The inhibition was reversed by raising the concentration of certain divalent metal ions, calcium in particular in the basal growth medium. The nature of the inhibition was studied. Heme and other porphyrins related to hemoglobin stimulated the growth of Euglena gracilis in light. Implications of these findings are discussed.