Quantitative and compositional changes in monogalactosyl and digalactosyl diglycerides during light-induced formation of chloroplasts in Euglena gracilis

The formation of chloroplasts in dark-grown cells of Euglena gracilis was induced by exposing the cells to constant illumination. Following a lag, the cells accumulated chlorophyll and galactosyl diglycerides simultaneously at almost linear rates. The monogalactosyl diglyceride content rose from approximately 2 micromoles in 100 mg of dark-grown cells to 27 micromoles in fully green cells; the digalactosyl diglyceride content increased from 1 micromole to 11 micromoles. The digalacto compounds increased more rapidly than the monogalacto compounds at first, but their rate of accumulation began to diminish long before greening of the cell was complete. The sole exception was the digalactosyl diglyceride fraction that contained hexadecadienoic (16:2) fatty acid. This fraction increased continuously during greening. As accumulation of the digalacto compounds diminished, that of the monogalacto compounds increased. Towards the end of greening, the major fatty acids were 16:2, 16:3, 16:4, 18:2, and 18:3 in the monogalacto and 16:2 in the digalacto compounds. The results of this study suggest that monogalactosyl and digalactosyl diglycerides that contain particular fatty acid components have a function in the assembly of chloroplasts.     

The bacterial-like lactate shuttle components from heterotrophic Euglena gracilis

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD(+)-independent d-lactate dehydrogenase (d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD(+)-dependent LDH specific for the generation of d- and l-lactate (d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K(+) or NH(4)(+). Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.     

Interactions between photoautotrophic and heterotrophic metabolism in photoheterotrophic cultures of Euglena gracilis

Interactions between photoautotrophic and heterotrophic metabolism in photoheterotrophic culture of Euglena gracilis were studied. Under a low light supply coefficient, these two metabolic activities seem to proceed independently. The cell growth rate in photoheterotrophic culture was about the sum of the growth rates in pure photoautotrophic and heterotrophic cultures. However under a high light supply coefficient, both photoautotrophic and heterotrophic (glucose assimilation) metabolic activities were inhibited, resulting in a low photoheterotrophic growth rate. The photoheterotrophic culture was more sensitive to photoinhibition compared to the pure photoautotrophic culture. Inhibition of glucose assimilation in the photoheterotrophic culture was due to both direct and indirect (through photosynthesis) effects of high light intensity. Cell growth, glucose assimilation and alpha-tocopherol content of the cells were higher when ambient air was used for aeration than when a mixture of carbon dioxide and air was used. Even when photosynthesis was inhibited by addition of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea to photoheterotrophic culture, light stimulated alpha-tocopherol synthesis by E. gracilis.     

Preparative isolation of monogalactosyl and digalactosyl diglycerides by thin-layer chromatography

Monogalactosyl and digalactosyl diglycerides were separated from leaf and Chlorella vulgaris lipid extracts by thin-layer chromatography with Silica Gel G as the stationary phase and acetone-acetic acid-water as the mobile phase. Phospholipids are completely removed and with two-dimensional development can themselves be fractionated.     

Actin Gene Sequence from Euglena gracilis

ABSTRACT The full length coding sequence of the Euglena gracilis actin gene was determined by RT-PCR of Euglena gracilis mRNA. Conserved regions in the actin amino acid sequence were used as guides for the synthesis of degenerate primers. Sequence was obtained for 1.238 nucleotides, of which 1.131 were coding for 377 amino acids. Sequence comparisons showed a similarity with other actins of 56% to 80%. Even though most of the actin amino acid sequence was conserved, some regions showed high divergence, i.e. the DNase I-binding loop at the N-terminal region. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.     

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.     

Influence of the alternative respiratory pathway on the adenylate pools in heterotrophic Euglena gracilis

Etiolated Euglena gracilis Pringsheim, strain Z, were cultured in a lactate medium either in the presence of 2 μ M antimycin A for cells adapted to this inhibitor, or in the absence of antimycin A for controls. The adenylates (ATP, ADP and AMP) and the energy charge (EC) were followed during the growth of both types of cells. The effects of KCN, salicylhydroxamic acid (SHAM) and rotenone on the respiration and the adenylate pool, were investigated during the exponental and stationary phases. EC values of controls and antimycin-adapted cells were not significantly different during culture. In the logarithmic phase, EC of controls was unaffected by 3 m M SHAM, an inhibitor of the alternative pathway, but markedly decreased by 0.3 m M KCN, which inhibits the cytochrome pathway. In contrast, in antimycin-adapted Euglena , in which the cytochrome pathway was blocked, ATP content and EC were markedly lowered in the presence of SHAM but slightly increased by 0.3 m M KCN. The combination of the preceeding treatments, as well as 15 m M KCN alone, were deleterious for both types of cells, in the logarithmic and the late stationary phases. The data indicate that the energy level in Euglena was dependent on the alternative pathway when the cytochrome pathway was blocked. Such dependence could be explained by the engagement of the first rotenone-sensitive site of phosphorylation. Indeed, 50 μ M rotenone caused a similar relative decrease of oxygen consumption and ATP content in controls and in antimycin-adapted Euglena . In the absence of cytochrome respiration, the alternative pathway allowed electrons to flow through this first segment of the respiratory chain, and ATP production by the first site of phosphorylation.     

Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis

Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s_{20, w} at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.     

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.