Purification and biochemical characterisation of a glucose-6-phosphate dehydrogenase from the psychrophilic green alga Koliella antarctica

Psychrophilic organisms have evolved a number of modifications of cellular structures to survive in the cold environment; among them it is worth noting an increased efficiency of enzymes at lower temperatures. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was purified and characterised from the psychrophilic green alga Koliella antarctica (Trebouxiophyceae, Chlorophyta) from the Ross Sea (Antarctica). It was possible to isolate a single G6PDH using biochemical strategies; its maximum activity was measured at 35 °C, and the enzyme showed an E _a of 39.6 kJ mol^?1. This protein reacted with antibodies raised against higher plants plastidic isoforms. KaG6PDH showed peculiar kinetic properties, with a K _iNADPH value lower than $ K_{{{\text{mNADP}}^{ + } }} $ . Notably, catalytic activity was inactivated in vitro by DTT and chloroplastic thioredoxin f. These biochemical properties of G6PDH are discussed with respect to higher plant G6PDHs and the adaptation of K. antarctica to polar low-temperature environment.     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana

Wild type (WT), and nitrate reductase (NR)- and nitrite-reductase (NiR)-deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate-grown WT cells was very low in air, increased slightly after addition of nitrite (200 microM), but strongly under anoxia. Importantly, even completely NR-free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR-free cells. In contrast, NiR-deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.     

A comparison of the high-active and low-active form of nitrate reductase in synchronous Chlorella sorokiniana

Chlorella sorokiniana possesses two forms of nitrate reductase (EC 1.6.6.1.). One with low activity is present in cells at the end of the light-dark cycle, the other with high activity is present after 1 h of illumination. The two forms can be distinguished by gel electrophoresis, isopycnic centrifugation, assay of the partial reactions and their sensitivity to antibodies, respectively. These differences are discussed with respect to an effect of intracellular nitrate on the activation of nitrate reductase.Abbreviations NAR nitrate reductase - FMN flavine mononucleotide - MV methylviologen     

Temperature dependence of extracellular enzymes production by psychrotrophic and psychrophilic bacteria

The effect of different growth temperatures on the production of 11 extracellular enzymes was studied in nine cold-adapted bacterial strains isolated from various cold environments. Ten of these enzymes displayed temperature-dependent production. Five different temperature-related production patterns were identified, which depended on neither the type of strain, nor the nature of the enzyme.     

Composition of the sheath produced by the green alga Chlorella sorokiniana

AIMS: To investigate the chemical characterization of the mucilage sheath produced by Chlorella sorokiniana. METHODS AND RESULTS: Algal mucilage sheath was hydrolysed with NaOH, containing EDTA. The purity of the hydrolysed sheath was determined by an ATP assay. The composition of polysaccharide in the sheath was investigated by high-performance anion-exchange chromatography with pulsed amperometric detection. Sucrose, galacturonic acid, xylitol, inositol, ribose, mannose, arabinose, galactose, rhamnose and fructose were detected in the sheath as sugar components. Magnesium was detected in the sheath as a divalent cation using inductively coupled argon plasma. The sheath matrix also contained protein. CONCLUSIONS: It appears that the sheath is composed of sugars and metals. Mucilage sheath contains many kinds of saccharides that are produced as photosynthetic metabolites and divalent cations that are contained in the culture medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on chemical characterization of the sheath matrix produced by C. sorokiniana.     

Nitrate reductase and glutamine synthetase activities,nitrate and ammonia assimilation,in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.     

Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases

The functional and structural adaptations to temperature have been addressed in homologous chloride-dependent α-amylases from a psychrophilic Antarctic bacterium, the ectothermic fruit fly, the homeothermic pig and from a thermophilic actinomycete. This series covers nearly all temperatures encountered by living organisms. We report a striking continuum in the functional properties of these enzymes coupled to their structural stability and related to the thermal regime of the source organism. In particular, thermal stability recorded by intrinsic fluorescence, circular dichroism and differential scanning calorimetry appears to be a compromise between the requirement for a stable native state and the proper structural dynamics to sustain the function at the environmental/physiological temperatures. The thermodependence of activity, the kinetic parameters, the activations parameters and fluorescence quenching support these activity-stability relationships in the investigated α-amylases.     

O-Dealkylation of coumarin and resorufin ethers by unicellular green algae: kinetic properties of Chlorella fusca and Chlorella sorokiniana

O-Dealkylations of resorufin and coumarin ethers, mediated by microsomal cytochrome P450 mono-oxygenases from animals, plants and microorganisms, are shown here to be performed also by intact cells of the unicellular green algaeChlorella fusca andChlorella sorokiniana. The activity of theO-dealkylation of these ethers was up to tenfold higher withChlorella sorokiniana. Both algae dealkylated methyl-, ethyl-, and pentylethers of resorufin and coumarin. Dealkylation in vivo indicated efficient absorption of methoxy- and ethoxyresorufin, confirmed by the respective absorption kinetics. Piperonylbutoxide and 1-aminobenzotriazole, known inhibitors of plant and mammalian cytochrome P450s, significantly inhibited theO-dealkylase activity of both algal strains. The use of synchronized cultures of both algae revealed that efficiency ofO-dealkylation depends on the stage of the cell cycle: during the growth phase, theO-dealkylase activities increased more than proportional, and the distinct drop in activity during the last hours of the light period indicated the appearance of an endogenous substrate.     

The acquisition and accumulation of inorganic carbon by the unicellular green alga Chlorella ellipsoidea

Abstract. The uptake and accumulation of inorganic carbon has been investigated in Chlorella ellipsoidea cells grown at acid or alkaline pH. Carbonic anhydrase (CA) was detected in ceil extracts but not in intact cells and CA activity in acid-grown cells was considerably less than that in alkali-grown cells. Both cell types demonstrates low K_1/2 (CO₂) values in the range pH 7.0–8.0 and these were unaffected by O₂ concentration. The CO₂ compensation concentrations of acid- and alkali-grown cells suspended in aqueous media were not significantly different in the range of pH 6.0–8.0, but at pH 5.0, the CO₂ compensation concentrations of acid-grown cells (57.4cm³ m⁻³) were lower than those of alkali-grown cells (79.2cm³ m⁻³). The rate of photo-synthetic O₂ evolution in the range pH 7.5–8.0 exceeded the calculated rate of CO₂ supply two- to three-fold, in both acid- and alkali-grown cells, indicating that HCO₃⁻ was taken up by the cells. Accumulation of inorganic carbon was measured at pH 7.5 by silicone-oil centri-fugation, and the concentration of unfixed inorganic carbon was found to be 5.1 mol m⁻³ in acid-grown and 6.4mol m⁻³ in alkali-grown cells. These concentrations were 4.6- and 5.9-fold greater than in the external medium. These results indicate that photorespiration is suppressed in both acid- and alkali-grown cells by an intracellular accumulation of inorganic carbon due, in part, to an active uptake of bicarbonate.     

Positive correlation between superoxide dismutase and resistance to paraquat toxicity in the green alga Chlorella sorokiniana

Paraquat (10–30 μm) exerted a dose-dependent and light-dependent toxicity on Chlorella sorokiniana. Paraquat was also seen to increase the superoxide dismutase content of these cells and to cause the appearance of a new electrophoretically distinct isozyme. Cells grown in the absence of paraquat contained one manganese-superoxide dismutase and two iron-superoxide dismutases, while the paraquat-grown cells contained an additional manganese-superoxide dismutase. Cells which were grown in the presence of 25 μm Paraquat, and which therefore possessed elevated levels of superoxide dismutase, were resistant to 30 μm Paraquat, whereas control cells were bleached and killed by this level of Paraquat. Electron micrography and chemical analysis revealed that Paraquat decreased the starch content of the cells and caused a failure of dividing cells to separate. It appears that Paraquat increases the photoproduction of O₂^? in C. sorokiniana and that an increase in the cell content of superoxide dismutase is an adaptive response which provides protection against this herbicide.     

Singlet oxygen damages the function of Photosystem II in isolated thylakoids and in the green alga Chlorella sorokiniana

Photosynthesis Research - Singlet oxygen (1O2) is an important damaging agent, which is produced during illumination by the interaction of the triplet excited state pigment molecules with molecular...     

Comparative analysis of photosynthetic and respiratory parameters in the psychrophilic unicellular green alga <Emphasis Type="Italic">Koliella antarctica</Emphasis>, cultured in indoor and outdoor photo-bioreactors

Effect of temperatures and illumination of temperate winter on photosynthesis and respiration was studied in the psychrophilic microalgae, Koliella antarctica (Trebouxiophyceae). Outdoor and indoor algal cultures were compared. Photosynthetic as well as respiration rates increased as light and temperature increased, until 35 °C, more in outdoor than in indoor cells, in agreement with the calculated Q₁₀ values. K. antarctica showed important strategy mechanisms of adaption to the several temperature and light conditions. These significant photo-acclimation and thermo-acclimation abilities make it possible to cultivate Koliella for different uses, under less expensive outdoor conditions. Therefore, K. antarctica shows important strategy mechanisms of adaption to various temperature and light conditions; moreover, by varying the culture conditions, it is possible to modulate and optimize the growth and accordingly the biomass production. This is a very interesting point since it has been proved that this microalga is a promising potential source of functional ingredients, such as polyunsaturated fatty acids and carotenoids, suitable for industrial purposes.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Temperature dependence of nitrate reductase activity in marine phytoplankton: biochemical analysis and ecological implications

The temperature dependence of NADH:NR activity was examined in several marine phytoplankton species and vascular plants. These species inhabit divergent thermal environments, including the chromophytes Skeletonema costatum (12–15° C), Skeletonema tropicum (18–25° C), Thalassiosira antarctica (?2 to 4° C), and Phaeocystis antarctica (?2 to 4° C), the green alga Dunaliella tertiolecta (14–28° C), and the vascular plants Cucurbita maxima (20–35° C) and Zea mays (20–25° C). Despite the difference in growth habitats, similar temperature response curves were observed among the chromophytic phytoplankton, with temperatures optimal for NR activity being between 10–20° C. In contrast, the chlorophyll b‐containing alga and vascular plants exhibited optimal temperatures for NR activity above 30° C. Such dramatic differences in NR thermal characteristics from the two taxonomic groups reflect a divergence in NR structure that may be associated with the evolutionary diversification of chromophytes and chlorophytes. Further, it suggests a potential contribution of the thermal performance of NR to the geographic distributions, seasonal abundance patterns, and species composition of phytoplankton communities. NR partial activities, which assess the individual functions of Mo‐pterin and FAD domains, were evaluated on NR purified from S. costatum to determine the possible causes for high temperature (>20° C) inactivation of NR from chromophytes. It was found that the FAD domain and electron transport among redox centers were sensitive to elevated temperatures. S. costatum cells grown at 5, 15, and 25° C exhibited an identical optimal temperature (15° C) for NADH:NR activity, whereas the maximal NR activity and NR protein levels differed and were positively correlated with growth temperature and growth rate. These findings demonstrate that thermal acclimation of NO₃^? reduction capacity is largely at the level of NR protein expression. The consequences of these features on NO₃^? utilization are discussed.     

Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca

[Fe]-hydrogenases are redoxenzymes that catalyze the reversible reduction of protons to hydrogen. Hydrogenase activity was observed in a culture of the unicellular green alga Chlorella fusca after an anaerobic incubation, but not in the related species Chlorella vulgaris. Specific polymerase chain reaction (PCR) techniques lead to the isolation of the cDNA and the genomic DNA of a special type of [Fe]-hydrogenase in C. fusca. The functional [Fe]-hydrogenase was purified to homogeneity and its N-terminus was sequenced. The polypeptide sequence shows a high degree of identity with the amino acid sequence deduced from the respective cDNA region. Structural and biochemical analyses indicate that ferredoxin is the main physiological electron donor.