Uptake and incorporation of pyrimidines in Euglena gracilis

In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concentrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance -AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and -AIB is inhibited by cycloheximide in a different manner after 5–10 min.Abbreviations Barb barbituric acid - 5-BrU 5-bromouracil - Cyd cytidine - Cyt cytosine - DHU dihydrouracil - dUrd deoxyuridine - dThd thymidine - 5-FU 5-fluorouracil - Ora orotic acid - Thy thymidine - Ura uracil - Urd uridine - CHI cycloheximide - -AIB -aminoisobutyric acid Dedicated to Prof. Dr. Dr. h.c. mult. K. Mothes on the occasion of his 75th birthday     

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.     

Effects of increased salinity on gravitaxis in Euglena gracilis

The unicellular freshwater flagellate Euglena gracilis regulates its position in the water column by means of phototactic and gravitactic behavior. Recent experiments have revealed that the cells switch between negative and positive gravitaxis depending upon environmental stimuli such as solar radiation. In this study, the effect of increased salinity on gravitaxis in Euglena gracilis was investigated. In some experiments it was found that salt concentrations up to 5 gL-1 (in some experiments 10 gL-1) increased the motility, velocity and precision of negative gravitactic orientation. Higher salt concentrations decreased all these parameters. At concentrations of about 15 gL-1, cells which did not become immobile, switched from negative to positive gravitaxis. Positive gravitaxis persisted for several hours or even days when the cells were transferred back to standard culture medium. Most of the cells in cultures exposed to salt concentrations above 20 gL-1 lost their motility (partial formation of palmella stages) but recovered when transferred back to standard medium or de-ionised water. Post recovery, the cells showed pronounced positive gravitaxis. Additional investigations on the pigmentation, revealed that the cells showed a complete loss of a carotenoid shoulder in the spectrum, which reappeared when the cells were brought back to standard medium.     

Transfer RNA genes of Euglena gracilis chloroplast DNA

Summary Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2–6 genes. None of the known genes contains introns, nor codes for the 3-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system

The studies described indicate that the UV bleached mutant, Euglena gracilis W₃BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplastic mutant, E. gracilis W₃BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W₃BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W₃BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.     

Photosensory transduction in the flagellated alga,Euglena gracilis

Euglena were cultured under 3 W m^-2 constant white light. In culture medium, cells show immediate and long lasting step-down photophobic responses and photoaccumulation behavior to blue light if dim red light-adapted for 30 min. However, if cells are suspended in buffered, saltcontaining solutions (adaptation buffers), strong step-down photobehavior and photoaccumulation responses are not observed for several hours. These behaviors gradually increase in strength to reach a maximum after 6–12 h; after which a stable response is maintained. The relative rates of appearance and the relative strengths of the responses are influenced by the concentrations of Ca²⁺ and K⁺, but not H⁺ or Na⁺ ions, in the adaptation buffers. Expression of the stepdown photobehavior thus requires that the cells adapt to the chemical environment in which they are suspended.Abbreviations Hepes N-2-hydroxypiperazine-N-2-ethanesulfonic acid - Mes 2(N-morpholino)-ethanesulfonic acid - Pipes piperazine-N,N-bis (2-ethanesulfonic acid) - Taps tris(hydroxymethyl) methylaminopropanesulfonic acid This work was supported, in part, by grant No. PCM-79-05320 from the U.S. National Science Foundation to B.D.     

Photosensory transduction in Euglena gracilis: Effect of some metabolic drugs on the photophobic response

We have investigated the effect of some metabolic drugs, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,4-dinitrophenol (DNP), sodium azide (NaN₃), on the photobehavior of single cells of Euglena gracilis, in order to clarify the relevance of different metabolic pathways in the process of photoperception and sensory transduction in this alga. The results obtained show that the photophobic response of Euglena is not affected by the action of these drugs. This suggests that neither the photosynthetic process nor oxidative phosphorylation play a significant role in the phenomenon of photosensory transduction in Euglena.List of Abbreviations DNP 2,4-dinitrophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI Photosystem I - PSII Photosystem II     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.     

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).     

Light dependency of resistance to ionizing radiation in Euglena gracilis

The resistance of Euglena gracilis strains Z (wild type) and SM-ZK (chloroplast-deficient mutant) to ionizing radiation was investigated. The colony forming ability of E. gracilis strain Z was higher than that of strain SM-ZK after 60Cogamma-irradiation. For both strains, the resistance of light-grown cells was higher than that of dark-grown cells, suggesting that the light conditions during culture contribute to the radiation resistance of E. gracilis. The comet assay showed that the ability of rejoining DNA double-strand breaks (dsb) was much higher in the light-grown cells. These results suggest that E. gracilis possesses a light-induced repair system to cope with DNA dsb.     

Indications for acceleration-dependent changes of membrane potential in the flagellate Euglena gracilis

Summary. The effects of the calcium sequester EGTA on gravitactic orientation and membrane potential changes in the unicellular flagellate Euglena gracilis were investigated during a recent parabolic-flight experiment aboard of an Airbus A300. In the course of a flight parabola, an acceleration profile is achieved which yields subsequently about 20 s of hypergravity (1.8 g _n), about 20 s of microgravity, and another 20 s of hypergravity phases. The movement behavior of the cells was investigated with real-time, computer-based image analysis. Membrane potential changes were detected with a newly developed photometer which measures absorption changes of the membrane potential-sensitive probe oxonol VI. To test whether the data obtained by the oxonol device were reliable, the signal of non-oxonol-labelled cells was recorded. In these samples, no absorption shift was detected. Changes of the oxonol VI signals indicate that the cells depolarize during acceleration (very obvious in the step from microgravity to hypergravity) and slightly hyperpolarize in microgravity, which can possibly be explained with the action of Ca-ATPases. These signals (mainly the depolarization) were significantly suppressed in the presence of EGTA (5 mM). Gravitaxis in parallel was also inhibited after addition of EGTA. Initially, negative gravitaxis was inverted into a positive one. Later, gravitaxis was almost undetectable. Correspondence and reprints: Department of Plant Ecophysiology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Federal Republic of Germany.     

Polarotaxis,gravitaxis and vertical phototaxis in the green flagellate,Euglena gracilis

A fully automatic computer-controlled video analysis system has been used to study the movement of the green unicellular flagellate, Euglena gracilis in a horizontal or vertical cuvette. In darkness, in the absence of gaseous gradients, most cells swim straight upwards. While in a horizontal cuvette the transition between positive and negative phototaxis is found at about 1.5 W m^-2, an excess of 30 W m^-2 is required to reverse the upward swimming (due to the combined stimulus of negative gravitaxis and positive phototaxis) in a vertical cuvette. By studying the swimming direction in horizontal and vertical cuvettes in polarized light irradiated from above or from the side, respectively, the dichroic orientation of the photoreceptor molecules can be determined in three dimensions with respect to the axes of the cell: In a horizontal cuvette, in a linearly polarized beam from above, the cells orient predominantly at an angle of about 30° clockwise off the electric dipole transition moment as seen from above. The behavior in a vertical cuvette with polarized light entering from above indicates that the photoreceptor pigments are dichroically oriented 60° counterclockwise from the flagellar plane (seen from the front end of the cell). Experiments with horizontal polarized light indicate that the photoreceptor transition moment deviates 25° clockwise off the long axis of the cell.Abbreviation PFB paraflagellar body Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Effects of acidity on the growth of two Euglena species

Our study separates the effects of elevated protons (at pH <3) and elevated metals (Al, Cd, Cu, Fe, Ni, Zn) on the growth of E. mutabilis Schmitz, a pioneering phototroph in acid mine drainage (AMD) and E. gracilis Klebs, a closely-related species rarely found in severely AMD-impacted sites. Both species were acid tolerant, growing optimally at pH 2.5–7. At pH values typical of AMD (pH 2.5–4) in the absence of elevated metals, E. gracilis outcompeted E. mutabilis (growth rates of 1.0 and 0.8 div d^–1, respectively). Relative metal toxicities were evaluated based on the Effective Exposure causing 50% growth reduction (= EE50). With total metal additions similar to AMD levels, E. mutabilis demonstrated significantly greater tolerance to all metals, except Cu. E. gracilis showed two-fold higher tolerance to Cu²⁺ than E. mutabilis (EE50 of 91.6 vs. 45.7 pmol cell^–1). The EE50 for Zn²⁺ was similar for both species (368 pmol cell^–1 for E. gracilis and 423 pmol cell^–1 for E. mutabilis). With Cd and Ni, E. mutabilis tolerated an order of magnitude higher exposure than E. gracilis(EE50 of 1.6 vs. 0.2 pmol Cd²⁺ cell^–1; EE50 of 942 vs. 87 pmol Ni²⁺ cell^–1). Al and Fe were tolerated at high total metal concentrations (up to 100 mM) by E. mutabilis, but toxicity was evident with E. gracilisat much lower levels. E. mutabilis grew at double the Al³⁺ exposure tolerated by E. gracilis (EE50 of 398 vs. 188 pmol Al³⁺ cell^–1). There was an 18-fold difference in Fe tolerance levels between E. mutabilis and E. gracilis with EE50s of 8773 and 502 pmol Fe²⁺ cell^–1, respectively. We conclude that differential metal tolerance, particularly to Fe²⁺, accounts for the mutually exclusive distribution of E. gracilis and E. mutabilis in AMD-impacted habitats.     

Constitutive overexpression of immunoidentical forms of PCP-induced Euglena gracilis CYP-450

Environmental pollutants are classically associated with increased drug metabolism. In this report, antibodies that are able to detect mammalian CYP proteins, namely the CYP1A1, CYP1A2, CYP2B1/B2, and CYP3A4 proteins, were used to investigate the expression of CYP-related proteins in Euglena gracilis (EG) cells under normal and PCP-treated conditions and in a EG-cell line adapted to PCP. Compared to normal conditions, the presence of PCP in the culture medium induced elevated levels of EG CYP-like proteins. With the exception of CYP3A4, this overexpression was correlated with expression of additional forms of CYP proteins having, respectively, the same molecular weight but slightly different pIs. Even in EG cells which had lost their PCP-adapted property after having been cultured without PCP, these additional forms were continuously expressed. This observation raised the question about the definition of a biomarker of pollution.     

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Summary. Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10mM MnSO₄. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion–tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.Correspondence and reprints: Dipartimento delle Risorse Naturali e Culturali, Università degli Studi di Ferrara, Corso Porta Mare 2, 44100 Ferrara, Italy.     

Photoreception inEuglena gracilis: Fluence-rate and time dependence of photoaccumulation and photodispersal

The fluence-rate and time dependence for photoaccumulation and photodispersal ofEuglena gracilis was measured for the wild-type strain and three white mutants. For wavelengths of 453 or 463 nm the threshold for photoaccumulation was close to 6×10⁻²Wm⁻². Photoaccumulation increased steadily with time and reached a maximum after about 4 hr. Red light elicited substantial photoaccumulation in the wild type and photodispersal in the white, non-photosynthetic mutant 1224-5/9f. The chromophore mediating the red-light response needs to be a non-photosynthetic pigment which remains presently unidentified. A whiteEuglena mutant, FB, which had retained a reduced stigma and a paraflagellar body, showed weak photoaccumulation. Two white mutants, 1224-5/1f and 1224-5/9f, both of which lacked the stigma and positive phototaxis, displayed during the first 90 min of irradiation photodispersal; after longer irradiations they showed instead photoaccumulation. These results contradict a widely held belief that the presence of a stigma represents a stringent requirement for photoaccumulation. Our results imply that phototaxis is not a prerequisite for photoaccumulation. Exogenous flavins and 5,10-methenyl-tetrahydrofolate (MTHF) influenced in a wavelength-dependent manner photoaccumulation and photodispersal. In the wild type FAD and riboflavin (RB) caused at 453 nm an increase of the responsiveness for photoaccumulation. The photoaccumulation of the white mutant FB, was sensitized by FMN and FAD. In the white mutant 1224-5/9f exogenous flavins lowered the threshold for photodispersal. FMN, which absorbs only blue light, altered also the responsiveness to red light: in the wild type FMN reduced photoaccumulation and in the white mutant 1224-5/9f it reduced photodispersal.