Isolation and characterization of thiosulfate reductases from the green alga Chlorella fusca

Thiosulfate-reductase activity (TSR) measured as sulfide release from thiosulfate was detected in crude extracts of Chlorella using dithioerythritol (DTE) as electron donor. Purification of this activity by ammonium-sulfate precipitation between 35% and 80% followed by Sephadex G-50 gel filtration, diethylaminoethyl-cellulose chromatography, and gel filtration on Biogel A 1.5 M led to four distinct proteins having molecular weights of: TSR I, 28000; TSR II, 26500; TSR III_a, 55000; TSR III_b, 24000 daltons. These thiosulfate reductases were most active with DTE; the monothiols glutathione, l-cysteine, and -mercaptoethanol had little activity towards this system. The following pH optima were obtained: for TSR I and TSR II, 9.0; for TSR III_a, 8.5; and for TSR III_b, 9.5. The apparent-K_m data for DTE and thiosulfate were determined to: TSR I, 0.164 mmol·l^-1 and TSR II, 0.156 mmol·l^-1; K_mDTE TSR I, 1.54 mmol·l^-1 and TSR II 1.54 mmol·l^-1. The thiosulfate reductases III_a and III_b were further stimulated by addition of thioredoxin. All TSR fractions catalyzed SCN formation from thiosulfate and cyanate and thus had rhodanese activity; this activity, however, could only be detected in the presence of thiols.Abbreviations DTE dithioerythritol - TSR thiosulfate reductase Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The dependence of the cytoplasmic pH in aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris on the pH of the medium as determined by 31P in vivo NMR spectroscopy

The pH in the cytoplasm of aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris was determined in dependence on the pH of the external medium, which was varied between pH 3 and pH 10. In aerobic cells of both species the cytoplasmic pH is maintained at a value above 7.2 even at an external pH of 3 and below 7.8 at an external pH of 10. In anaerobic cells the cytoplasmic pH shows linear dependence on external pH in the range of pH 6 to 9 (cytoplasmic pH 6.9 to 7.2), while below an external pH of 6 cytoplasmic pH is maintained at about 6.5.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - EDTA Ethylendiaminetetraacetic acid - MES 2-(N-Morpholino)-ethanesulfonic acid - MOPSO 3-(N-Morpholino)-2-hydroxy-propanesulfonic acid - NMR Nuclear Magnetic Resonance - pH cyt cytoplasmic pH - pH ex external pH - PIPES Piperazine-N,N-bis(2-ethanesulfonic acid) - PP_i Pyrophosphate - PP₁, PP₂, PP₃ 1st, 2nd, 3rd phosphate group of polyphosphates - PP₄ core phosphate groups of polyphosphates - TRIS Tris-hydroxymethyl-aminomethane     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Heterologous expression of cDNAs encoding Chlorella sorokiniana NADP-specific glutamate dehydrogenase wild-type and mutant subunits in Escherichia coli cells and comparison of kinetic and thermal stability properties of their homohexamers

Full-length cDNAs encoding the - and -subunits and a truncated mutant subunit of the Chlorella sorokiniana NADP-GDH isozymes were constructed and expressed in Escherichia coli cells. The kinetic and thermal stability properties of the resultant homohexamers were examined. The electrophoretic mobility of the recombinant - and -subunits was identical to that of the native subunits as determined by immunoblotting. The homohexamers were purified by anion-exchange and gel-filtration chromatography. The - and -homohexamers that were synthesized in the bacterial cells were shown to have similar Michaelis constants for their substrates as previously shown after synthesis in C. sorokiniana cells (Bascomb and Schmidt, 1987). The homohexamer synthesized in the bacterium was allosteric with respect to NADPH but to a lesser degree than when isolated from the alga. The mutant homohexamer was composed of subunits that were truncated by 40 amino acids at their N-termini. This mutant isozyme was kinetically similar to the larger, anabolic -homohexamer, but it did not display the allosteric response to NADPH shown by the -homohexamer. The three isozymes had significant thermal tolerance and were stable at 50 °C. The temperature optimum for catalytic activity for the - and -homohexamers was 60 °C, and 65 °C for the 40N homohexamer. This study demonstrated that most of the kinetic properties of the Chlorella sorokiniana NADP-GDH isozymes were retained after their synthesis in a heterologous system, and that the distinctive N-terminal domains of these isozymes have dramatic effects on their biochemical characteristics.     

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.     

Alkylsulfonic acids and some S-containing detergents as sulfur sources for growth of Chlorella fusca

Chlorella fusca can utilize the following substances as sole sulfur sources for growth: C₁ to C₈ n-alkane-1-sulfonates, linear alkylbenzenes sulfonates (LAS), -sulfonated fatty acid esters, polyethylene glycol sulfate and alkylsulfates. Good sulfur sources are alkylsulfonic acids, which are comparable to sulfate. Ethanesulfonic acid was used for comparison of the growth on sulfate and on a sulfonic acid, because best growth was achieved on this C₂-sulfonic acid.Growth data of Chlorella on the enviromental important detergents linear alkylbenzene sulfonic acids, -sulfonated fatty acid methylester, Texapon and Sulfopon are presented. So far only microorganisms have been discussed as a source for degradation of sulfonic acids and detergents. It is suggested that green algae could be of similar importance for the biodegradation of these compounds.Abbreviations LAS Linear alkylbenzene sulfonate - ES -sulfonated fatty acid methylester - DTE dithiocrythritol     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.     

Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca

Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO₂), but they did not if supplied with air containing 5% CO₂. After a change from high to low CO₂ concentration in the gas stream, nitrite excretion started immediately. After an increase in CO₂ concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.Abbreviations FMN riboflavin 5-phosphate - GS glutamine synthetase - NIR nitrite reductase - NR nitrate reductase     

Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca

Isolated membrane fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of l-cysteine to l-cystine. The pH-optimum of this reaction was determined to be around 8–8.5 and a stoichiometry of 4 SH-groups oxidized for one O₂ consumed was obtained. This thiol-oxidation system was specific for D-and l-cysteine; Dl-homocysteine and cysteamine were oxidized at about half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were not oxidized by these membrane fractions. The apparent K_m for l-cysteine was determined as 3.3 mmol l^-1. Rates of 200 mol cysteine oxidized mg^-1 chlorophyll h^-1 were normally obtained. Extremely high rates of oxygen uptake were measured using l-cysteine methyl ester and l-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electron-transport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N-disalicyliden-1,3-diaminopropane 5,5-disulfonic acid, ethylenediaminetetraacetic acid, high KCN levels and by the buffers, N-[2-hydroxyl-1,1-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by O₂ (dark inactivation).Abbreviation DTNB 5,5-dithio-bis(-2-nitrobenzoic acid). Ellmann reagent     

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     

The relationship between heat-stress and photobleaching in green and blue-green algae

Two characteristic temperatures were identified from measurements of the temperature dependence of O₂ evolution by Chlorella vulgaris and Anacystis nidulans: T₁, the threshold temperature for inhibition of O₂ evolution under saturating light conditions, and T₂, the upper temperature limit for O₂ evolution. Measurement of delayed light emission from photosystem II (PSII) showed that it passed through a maximum at T₁ and was virtually eliminated on heating the samples to T₂. Related changes were observed in low-temperature (77K) fluoresence emission spectra. Heat-stress had little effect on the absorption properties of the cells at temperatures below T₁ but incubation at higher temperatures, particularly under high-light conditions, resulted in extensive absorption losses. An analysis of these measurements suggests that this increased susceptibility to photobleaching is triggered by an inhibition of the flow of reducing equivalents from PSII that normally serves to protect the light-harvesting apparatus of the cells from photo-oxidation. Adaptation to higher growth temperatures resulted in increases in the values of T₁ and T₂ for Anacystis nidulans but not for Chlorella vulgaris.Abbreviations PSI photosystem I - PSII photosystem II - Chl a chlorophyll a - Chl b chlorophyll b - DCMU 3-(3 4 dichlorophenyl)-11-dimethylurea - PC plastocyanin - APC allophycocyanin CIW-DPB Publication No. 887.     

Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.     

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     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Growth of Chlorella sorokiniana in the presence of sulfite elevates cell content of superoxide dismutase and imparts resistance towards paraquat

1. Growth of Chlorella sorokiniana in the presence of 7.5 mM sulfite, which halved the growth rate while doubling the superoxide dismutase (SOD; EC 1.15.1.1) content per cell, rendered the cells resistant to the toxic effects of 30 M paraquat. 2. While increasing total SOD content, sulfite increased the relative amount of the H₂O₂-resistant manganese-containing SOD. 3. It appears that O₂ ^– may be involved in mediating the toxicity of SO₂ in this green alga.Abbreviations SOD superoxide, dismutase - FeSOD ironcontaining superoxide dismutase - MnSOD manganese-containing superoxide dismutase     

The role of endosymbiotic algae in photoaccumulation of green Paramecium bursaria

The endosymbiotic unit of Paramecium bursaria with Chlorella sp. photoaccumulates in white, blue-green, and red light (<700 nm), whereas alga-free Paramecia never do. The intensity of photoaccumulation depends on both the light fluence rate and the size of the symbiotic algal population. Photoaccumulation can be stopped completely with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. Hence the photosynthetic pigments of the algae act as receptors of the light stimulus for photomovement and a close connection must exist between photosynthesis of the algae and ciliary beating of the Paramecium.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.     

Identification of Chlorella viruses in Paramecium bursaria clones by pulse-field electrophoresis

The ciliates Paramecium bursaria contain endosymbiotic green algae Chlorella spp. in their cytoplasm. The algae isolated from P. bursaria are sensitive to large DNA-containing viruses of the family Phycodnaviridae. The type virus of this family is PBCV-1 (Paramecium bursaria Chlorella virus). Investigation of the total DNA of P. bursaria clones by pulse-field electrophoresis (PEGE) revealed a pronounced band on PEGE profiles of some P. bursaria clones; the band was formed by DNA molecules of approx. 300 kb. This band probably contained the DNA of Chlorella virus. Two approaches were used in the present work to confirm this hypothesis. Microbiological tests were used to scan a collection of P. bursaria clones for specific types of viruses; the 300-kb band was revealed only in the PEGE profiles of virus-containing clones. Blot hybridization of P. bursaria total DNA separated by pulse-field electrophoresis with the virus-specific probe revealed that the band under study was formed by the DNA of a Chlorella virus. Paramecium clones were shown to contain approx. 10⁵ copies of nonintegrated viral DNA.     

Flocculation of algae using chitosan

Flocculation of three freshwater algae, Spirulina,Oscillatoria and Chlorella, and onebrackish alga, Synechocystis, using chitosan was studiedinthe pH range 4 to 9, and chlorophyll-a concentrations inthe range 80 to 800 mg m^–3, which produces aturbidity of 10 to 100 nephelometric turbidity units (NTU) in water. Chitosanreduced the algal content effectively by flocculation and settling. Theflocculation efficiency is very sensitive to pH, and reached a maximum at pH7.0for the freshwater species, but lower for the marine species. The optimalchitosan concentration that is required to effect maximum flocculation dependedon the concentration of alga. Flocculation and settling were faster whenconcentrations of chitosan higher than optimal are used. The settled algalcellsare intact and live, but will not be redispersed by mechanical agitation. Thede-algated water may be reused to produce fresh cultures of algae.