Photosynthetic Euglena Gracilis Research Articles

Microaerophilia enhances heavy metal biosorption and internal binding by polyphosphates in photosynthetic Euglena gracilis

Euglena gracilis is a photosynthetic protist with proved ability to resist and remove toxic single heavy metals under different laboratory conditions. To assess these abilities under more realistic conditions, cells subjected to combinations of heavy metals in a defined culture medium as well as in field polluted enriched medium were analyzed. Cells were cultured in the presence of Cd2+plus Cu2+ or Cr(VI) plus Fe3+ under aerobiosis or microaerophilia. Under aerobic growth, Cd2+plus Cu2+ (50 + 80 μM) and Cr(VI) plus Fe3+ (2 + 1 mM) strongly inhibited cell growth by inducing severe oxidative stress. In contrast, under microaerophilia, higher resistance and heavy metal removal were attained. This was indicated by (i) substantial biomass production; (ii) greater activities of glutathione reductase, glutathione peroxidase and ascorbate peroxidase as well as greater amounts of intracellular glutathione and ascorbate; (iii) increased metal removal by biosorption (i.e. greater than 80% for Cd, Cr, and Fe) due to an enhanced biofilm synthesis. The fraction of intracellularly heavy metal accumulated was bound mostly by polyphosphates (polyP). Cells cultured under microaerophilia in heavy metal polluted wastewater and sediments media were also able to grow and remove Fe, Cr, Cu and Cd. It is concluded that microaerophilia favors, the resistance of photosynthetic Euglena gracilis to heavy metal toxicity, and its ability to remove such metals from polluted water and solid samples. This work provides the biochemical basis for designing genetically engineered microorganisms with enhanced efficiency in metal removal. Moreover, it brings forward the combination of physicochemical and biological techniques to profit recovery.

Lead accumulation in photosynthetic Euglena gracilis depends on polyphosphates and calcium.

Worldwide increasing levels of lead in water systems require the search for efficient ecologically friendly strategies to remove it. Hence, lead accumulation by the free-living algae-like Euglena gracilis and its effects on cellular growth, respiration, photosynthesis, chlorophyll, calcium, and levels of thiol- and phosphate-molecules were analyzed. Photosynthetic cells were able to accumulate 4627mg lead/kgDW after 5 days of culture with 200μMPb2+. Nevertheless, exposure to 50, 100 and 200μM Pb2+ for up to 8 days did not modify growth, viability, chlorophyll content and oxygen consumption/production. Enhanced biosynthesis of thiol molecules and polyphosphates, i.e. the two canonical metal ion chelation mechanisms in E.gracilis, was not induced under such conditions. However, in cells cultured in the absence of phosphate, lead accumulation and polyphosphate content markedly decreased, while culturing in the absence of sulfate did not modify the accumulation of this metal. In turn, the total amount of intracellular calcium slightly increased as the amount of intracellular lead increased, whereas under Ca2+ deficiency lead accumulation doubled. Therefore, the results indicated that E.gracilis is highly resistant to lead through mechanisms mediated by polyphosphates and Ca2+ and can in fact be classified as a lead hyperaccumulator microorganism.

Accumulation of zinc protects against cadmium stress in photosynthetic Euglena gracilis

To determine the interplay between zinc and cadmium on the heavy metal accumulation capacity of Euglena gracilis, the effects of increasing Zn2+ concentrations (13–65.4ppm or 200–1,000μM) were analyzed on growth; O2 consumption; photosynthesis; ascorbate (APX) and glutathione peroxidase (GPX) activities; chlorophyll a and b (Chl a+b) content; essential metals, thiol-metabolites and polyphosphates (polyPs) levels; as well as on zinc and cadmium accumulation capacities. Control cells (EgZn20; grown with 20μM Zn2+) showed a half-maximal inhibition of growth (IC50) of 1,700μM by external Zn2+. O2 consumption, and APX and GPX activities were unaltered by Zn2+ treatments. Cells cultured with 500 or 1,000μM Zn2+ showed photosynthesis impairment but normal Chl a+b contents. Zn2+ preconditioning increased the intracellular contents of zinc (25–54 times) and calcium (2–27 times); thiol-metabolites and polyPs were only marginally altered. The growth of cells preconditioned to 400μM Zn2+ (EgZn400 cells) was less susceptible to Cd2+ than that of EgZn20 cells, although no differences in photosynthesis and respiration were observed. In cells chronically grown with Zn2+, the cadmium accumulation capacity was unchanged or slightly increased in the same culture media with high Zn2+, and increased by 42–90% in media with 20μM Zn2+. The thiol-metabolites increased at similar levels in both EgZn20 and EgZn400 cells when further exposed to 200μM Cd2+ and polyPs were at high levels independently of Zn2+ or Cd2+ treatments. It was concluded that chronic exposure to high Zn2+ (1) was innocuous for E. gracilis at concentrations lower than 0.5mM and (2) promoted protection against Cd2+ toxicity and increased cadmium accumulation; and (3) these zinc effects involved GSH and polyPs metabolism and were associated with high intracellular zinc contents.

Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis

The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn(2+) or Cd(2+), phytochelatin2-phytochelatin4 (PC2-PC4) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC2 as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC2 synthesis with Km values of 14-22 mM for GSH and 1.6-2.5 μM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-(GS)2, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)2 over Cd-(GS)2 as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.

Sulfate uptake in photosynthetic Euglena gracilis. Mechanisms of regulation and contribution to cysteine homeostasis

BackgroundSulfate uptake was analyzed in photosynthetic Euglena gracilis grown in sulfate sufficient or sulfate deficient media, or under Cd2+ exposure or Cys overload, to determine its regulatory mechanisms and contribution to Cys homeostasis. ResultsIn control and sulfate deficient or Cd2+-stressed cells, one high affinity and two low affinity sulfate transporters were revealed, which were partially inhibited by photophosphorylation and oxidative phosphorylation inhibitors and ionophores, as well as by chromate and molybdate; H+ efflux also diminished in presence of sulfate. In both sulfate deficient and Cd2+-exposed cells, the activity of the sulfate transporters was significantly increased. However, the content of thiol-metabolites was lower in sulfate-deficient cells, and higher in Cd2+-exposed cells, in comparison to control cells. In cells incubated with external Cys, sulfate uptake was strongly inhibited correlating with 5-times increased intracellular Cys. Re-supply of sulfate to sulfate deficient cells increased the Cys, γ-glutamylcysteine and GSH pools, and to Cys-overloaded cells resulted in the consumption of previously accumulated Cys. In contrast, in Cd2+ exposed cells none of the already elevated thiol-metabolites changed. Conclusions(i) Sulfate transport is an energy-dependent process; (ii) sulfate transporters are over-expressed under sulfate deficiency or Cd2+ stress and their activity can be inhibited by high internal Cys; and (iii) sulfate uptake exerts homeostatic control of the Cys pool.

Chromium uptake, retention and reduction in photosynthetic Euglena gracilis

Photosynthetic Euglena gracilis grown with different K(2)CrO(4) concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl(3). Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K (m) of 1.1 mM, V (m) of 16 nmol (h x 10(7) cells)(-1), and K (i sulfate) of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K (m) = 0.83 mM, V (m) = 15.9 nmol (h x 10(7)cells)(-1) and K (i chromate) = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 x 10(7) cells at 1 mM K(2)CrO(4)) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 x 10(7) cells at 1 mM CrCl(3)). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.

Simultaneous Cd2+, Zn2+, and Pb2+ Uptake and Accumulation by Photosynthetic Euglena gracilis

The ability of Euglena gracilis to simultaneously remove and accumulate Zn2+, Cd2+, and Pb2+ from culture up- to media was evaluated. E. gracilis was able to remove up to 80% of the Cd2+ present in the medium when cultured with 20 or 50 microM CdCl2. Higher external Cd2+ concentrations increased Cd2+ accumulation per cell but decreased cell growth, thus decreasing the capacity of the cell culture to remove Cd2+. E. gracilis removed 70% to 80% of the Zn2+ present in the medium when cultured with 5 to 50 microM ZnSO4. Zn2+ did not affect Cd2+ removal capacity. E. gracilis was much less efficient in removing Pb2+ (<15%) when cultured with 100 or 200 microM Pb(NO3)2. Moreover, Pb2+ decreased the efficiency to remove Cd2+, but it did not affect Zn2+ removal. Cd2+ induced a generalized increase in the cellular thiol compounds, including phytochelatins, and Pb2+ had an additive effect only at 200 microM. Zn2+ did not stimulate phytochelatin synthesis. Cd2+ and Pb2+ colocated in the same cytosolic high-molecular-weight fraction. Because Pb2+ is a weak phytochelatin inducer, competition between Pb2+ and Cd2+ for transportation across the plasma membrane and binding to phytochelatins and other thiol compounds is proposed to explain the detrimental effects of Pb2+ on the Cd2+ removal capacity of E. gracilis.

Enhanced Heavy Metal Tolerance in Two Strains of Photosynthetic Euglena gracilis by Preexposure to Mercury or Cadmium

The effects of different concentrations of Hg, Cd, and Pb on cell growth, oxygen consumption, and production and contents of chlorophyll a and b of Z and B strains of Euglena gracilis was studied. Cell uptake of Cd and Pb was also determined. A greater cell density reached in the presence of Cd2+ correlated with a lower metal uptake capacity in B-strain cells. Cells of Z strain, preexposed to low concentrations of either Hg2+ (1.5 μM) or Cd2+ (50 μM) for several generations showed improved heavy metal tolerance (greater cell density and viability) together with similar metal uptake capacity. Because Cd2+ preadaptation but not Hg2+ pretreatment induced a significant increase in the content of acid-soluble thiols, the data suggest the existence of different mechanisms of heavy metal detoxification in Euglena. The fact that Z-strain cells showed a greater Cd2+ uptake under all the conditions assayed suggests that Z strain would be more suitable for bioremediation of polluted waters.

Osservazioni ultrastrutturali su particolari organelli polimembranosi in Euglena gracilis

Abstract Ultrastructural observations on polymembranous organelles in Euglena gracilis. Polymembranous organelles, similar to hypertrophied mitochondria, seen in the cytoplasm of bleached or photosynthetic Euglena gracilis cells are presented.