Alga Chlorella Fusca Research Articles

Structural and Stereochemical Studies of Hydroxyanthraquinone Derivatives from the Endophytic Fungus Coniothyrium sp

Four known hydroxyanthraquinones (1-4) together with four new derivatives having a tetralone moiety, namely coniothyrinones A-D (5-8), were isolated from the culture of Coniothyrium sp., an endophytic fungus isolated from Salsola oppostifolia from Gomera in the Canary Islands. The structures of the new compounds were elucidated by detailed spectroscopic analysis and comparison with reported data. The absolute configurations of coniothyrinones A (5), B (6), and D (8) were determined by TDDFT calculations of CD spectra, allowing the determination of the absolute configuration of coniothyrinone C (7) as well. Coniothyrinones A (5), B (6), and D (8) could be used as ECD reference compounds in the determination of absolute configuration for related tetralone derivatives. This is the first report of anthraquinones and derivatives from an isolate of the genus Coniothyrium sp. These compounds showed inhibitory effects against the fungus Microbotryum violaceum, the alga Chlorella fusca, and the bacteria Escherichia coli and Bacillus megaterium.

Bioactive Aromatic Derivatives from Endophytic Fungus, Cytospora sp

Two new benzyl gamma-butyrolactone analogues, (R)-5-((S)-hydroxy(phenyl)-methyl)dihydrofuran-2(3H)-one (1) and its 6-acetate (2), and a new naphthalenone derivative (8), together with eight additional known aromatic derivatives, (S)-5-((S)-hydroxy(phenyl)-methyl)dihydrofuran-2(3H)-one (3), (S)-5-benzyl-dihydrofuran-2(3H)-one (4), 5-phenyl-4-oxopentanoic acid (5), gamma-oxo-benzenepentanoic acid methyl ester (6), 3-(2,5-dihydro-4-hydroxy-5-oxo-3-phenyl-2-furyl)propionic acid (7), (3R)-5-methylmellein (9), integracins A (10) and B (11) were isolated from Cytospora sp., an endophytic fungus isolated from Ilex canariensis from Gomera. The structures of these compounds were elucidated by detailed spectroscopic analysis, comparison with reported data, and chemical interconversion. The absolute configurations of the new compounds (1, 2, 8) were established on the basis of optical rotation or CD spectra analysis. Preliminary studies showed antimicrobial activity of these compounds against the fungi Microbotryum violaceum, Botrytis cinerea and Septoria tritici, the alga Chlorella fusca, and the bacterium Bacillus megaterium.

Cameroonemide A: a new ceramide from Helichrysum cameroonense

From the extracts of all parts of the plant Helichrysum cameroonense, five compounds were isolated and identified. One of them, a ceramide, named cameroonemide A (1), is reported for the first time as a new natural product. Its structure was determined by comprehensive analyses of their 1D and 2D NMR and HR-EI-MS spectral data. The remaining four known compounds were identified by comparing their spectroscopic data with those reported in the literature as kaurenoic acid (2), 3-acetyloxykaurenoic acid (3), β-sitosterol (4), and β-sitosterol glucopyranoside (5). Preliminary studies showed that 3-acetyloxykaurenoic acid (3) inhibited the alga Chlorella fusca, while kaurenoic acid (2) showed strong antibacterial activity against Bacillus megaterium.

Ring B Aromatic Steroids from an Endophytic Fungus, Colletotrichum sp.

The new (22E,24R)-3-acetoxy-19(10-->6)-abeo-ergosta-5,7,9,22-tetraen-3beta-ol (1) and the known (22E,24R)-19(10-->6)-abeo-ergosta-5,7,9,22-tetraen-3beta-ol (2), two interesting ergosteroids with rare aromatized ring B, together with seven known derivatives, namely (22E,24R)-ergosta-5,7,22-trien-3beta-ol (3), (22E,24R)-ergosta-4,7,22-trien-3-one (4), (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (5), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (6), (22E,24R)-ergosta-7,22-dien-3beta,5alpha,6beta-triol (7), (22E,24R)-6-acetoxy-ergosta-7,22-dien-3beta,5alpha,6beta-triol (8), and (22E,24R)-3,6-diacetoxy-ergosta-7,22-dien-3beta,5alpha,6beta-triol (9), were isolated from Colletotrichum sp., an endophytic fungus isolated from Ilex canariensis from Gomera. The structures of these compounds were elucidated by detailed spectroscopic analysis, comparison with reported data, and chemical interconversion. The isolation of these metabolites not only displays a beautiful array of chemical diversity, but also gives insight into the biosynthetic interconnections. Preliminary studies showed antimicrobial activity of these compounds against the fungus Microbotryum violaceum, the alga Chlorella fusca, and the bacteria Escherichia coli and Bacillus megaterium.

RELATIONSHIPS AMONG SPECIFIC GROWTH, SECRETED ACID PHOSPHATASE, INTRACELLULAR AND TOTAL INORGANIC PHOSPHORUS CONCENTRATION IN CHLORELLA FUSCA CULTURED UNDER VARIOUS LEVELS OF NA2HPO4

The relationships among specific growth rates, intracellular inorganic phosphate (Pi) and total phosphorus concentrations and specific activities of acid phosphatase (acPase) were studied on the green unicellular alga Chlorella fusca at 1.0, 15.0 and 30.0 µM Na2HPO4. The specific growth, the intracellular Pi and total phosphorus concentration increased with increasing Na2HPO4 levels. The acid phosphatase activity was inversely correlated with the specific growth rate, the total P concentrations and the intracellular inorganic concentrations. The P-deficiency induction of acPase activity is related to a decrease in P-availability in Chlorella fusca.

EFFECT OF TEMPERATURE ON THE ACTIVITY OF ACID PHOSPHATASES ISOLATED FROM CHLORELLA FUSCA, CLADOPHORA GLOMERATA AND PHORMIDIUM FAVEOLARUM AND THERMAL STABILITY OF THE ENZYMES

Temperature effect has been investigated in this study on the phosphatases isolated from three algae representing different groups; a unicellular freshwater green alga Chlorella fusca, a filamentous green macroalga Cladophora glomerata and a filamentous freshwater blue green microalga, Phormidium faveolorum. The temperature dependence of the enzyme activity could be summarized as that 60 oC was the optimum temperature for the enzyme activity in Cladophora glomerata as well as Chlorella fusca whereas Phormidium faveolorum had an optimum temperature of 70 oC for both isolated enzyme fractions, the cell wall-bound and the secreted extracellular. The thermal behavior of the phosphatases isolated from the tested algae showed toleration to the thermal activation when p-nitrophenyl phosphate (pNPP) was utilized as a substrate which has been investigated to be itself is not sensitive to high temperatures. The two fractions of the enzyme isolated from the algae exhibited high stability at high temperatures even above 80 oC

Diversity of Antimicrobial Pyrenophorol Derivatives from an Endophytic Fungus, Phoma sp.

AbstractPyrenophorol (1) and (–)‐dihydropyrenophorin (3), two known macrodiolides, were isolated together with four new analogues (2, 4–6) and three ring‐opened derivatives (7–9) from Phoma sp., an endophytic fungus isolated from Lycium intricatum from Gomera. The structures of the new compounds were elucidated by detailed spectroscopic analysis, comparison with reported data, and chemical interconversion. The absolute configurations were determined by chemical correlations and a modified Mosher's method. The diversity of these seven newly discovered metabolites not only extends the pyrenophorol macrocyclic family, but also gives insight into the biosynthetic interconnections, in particular by isolation of the open‐chain precursors. Preliminary studies showed antimicrobial activity of these compounds against the fungus Microbotryum violaceum, the alga Chlorellafusca, and the bacteria Escherichia coli and Bacillus megaterium.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

New Mono‐ and Dimeric Members of the Secalonic Acid Family: Blennolides A–G Isolated from the Fungus Blennoria sp.

Blennolides A-G (2-8), seven unusual chromanones, were isolated together with secalonic acid B (1) from Blennoria sp., an endophytic fungus from Carpobrotus edulis. This is the first reported isolation of the blennolides 2 and 3 (hemisecalonic acids B and E), the existence of which as the monomeric units of the dimeric secalonic acids had long been postulated. A compound of the proposed structure 4 (beta-diversonolic ester) will need to be revised, as its reported data do not fit those of the established structure of blennolide C (4). Other monomers, the blennolides D-F (5-7) seem to be derived from blennolides A (2) and B (3) by rearrangement of the hydroaromatic ring. The heterodimer 8, composed of the monomeric blennolide A (2) and the rearranged 11-dehydroxy derivative of blennolide E (6), extends the ergochrome family with an ergoxanthin type of skeleton. The structures of the new compounds were elucidated by detailed spectroscopic analysis and further confirmed by an X-ray diffraction study of a single crystal of 2. The absolute configurations were determined by TDDFT calculations of CD spectra, including the solid-state CD/TDDFT approach. Preliminary studies showed strong antifungal and antibacterial activities of these compounds against Microbotryum violaceum and Bacillus megaterium, respectively. They were also active against the alga Chlorella fusca and the bacterium Escherichia coli.

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.

Non-photochemical quenching of chlorophyll fluorescence in Chlorella fusca acclimated to constant and dynamic light conditions.

Non-photochemical quenching of chlorophyll fluorescence (NPQ) involves dissipation of light energy in the photosynthetic apparatus via a number of physiologically distinct processes. The relationships among NPQ, the (de)epoxidation state of the xanthophyll cycle pigments and state transitions was studied in the green alga Chlorella fusca, acquired from six differently light-acclimated continuous cultures. A 10 h light and 14 h darkness, periodicity was obeyed in all cultures. Three cultures received a high total daily irradiance, three others a low one. High and low irradiances were each dosed in three different modes at constant supply, with sine shape intensity modulation, or as a sine with superimposed oscillations. In the constant supply mode, but not for the sine and oscillating modes, high-light rendered a three-fold higher xantophyll cycle pigment content than low-light. Dynamic interconversion of xantophyll cycle pigments was restricted to high-light cultures. NPQ followed the kinetics of the light supply mode and was highest in high light cultures. In low-light cultures, NPQ correlated mainly to state transitions. These observations were supported by experiments with dithiothreithol-treated samples. The relative impact of xantophyll cycle operation and state transitions on NPQ in green algae from different light climates will be discussed with reference to higher plants.

Characterisation of cytochrome c 6 from Chlorella fusca

A c-type monohaem, cytochrome c6was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an apparent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 20.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-terminal sequence shows high identity with other green algae cytochromes c6. The mid-point redox potential is about +350 mV between pH 5 and 9. The ferric and ferrous forms, and their pH equilibria, have been studied using visible, CD and EPR spectroscopies. The visible spectrum of the reduced cytochrome c6is typical of a c-type haem protein, with maxima at 274 nm, 318 nm (δ-peak), 416 nm (γ-peak), 522 nm (β-peak), 552–553 nm (α-peak). A 690 nm band, characteristic of a haem Met-His axial coordination of the haem group, is present in the oxidized form. At high pH values (≥ 8), cytochrome c6undergoes an alkaline transition, with a pKa of 8.7. Between pH 3 and 9 the EPR spectrum is dominated by two rhombic species, with g-values at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert with a pKaof 4. CD spectrum of Chlorella fusca cytochrome c6shows that the proteins must be mainly built up by α-helices. Even though there are similarities between Chlorella fusca cytochrome c6and that isolated from Monoraphidium braunii, no cross-reactivity with the antibodies raised against the Chlorella fusca cytochrome has been detected for the protein from Monoraphidium braunii.

Pigment-pigment interactions and secondary structure of reconstituted algal chlorophyll a/b-binding light-harvesting complexes of Chlorella fusca with different pigment compositions and pigment-protein stoichiometries

Earlier we have shown by in vitro reconstitution experiments that the pigment composition of the chlorophyll alb-binding light-harvesting complex of the green alga Chlorella fusca could be altered in a relatively broad range (Meyer and Wilhelm 1993). In this study we used these reconstituted complexes of different pigment loading to analyze the excitonic interactions between the pigment molecules and the secondary structure by means of circular dichroism spectra in the visible and the far UV spectral regions, respectively. We found that, in contrast to the expectations, the pigment composition and pigment content hardly affected the circular dichroism spectra in the visible spectral region. Reconstituted complexes, independent of their pigment composition, exhibited the most characteristic circular dichroism bands of the native light-harvesting complex, even if one polypeptide bound only 3 chlorophyll a, 3 chlorophyll b and 1-2 xanthophyll molecules. Full restoration of the protein secondary structure, however, could not be achieved. The α-helix content depended significantly on the pigment composition as well as on the pigment-protein ratio of the reconstituted complexes. Further binding of pigments resulted in restoration of the minor excitonic circular dichroism bands, the amplitudes of which depended on the pigment content of the reconstituted complexes. These data suggest that in the reconstitution of light-harvesting complexes a 'central cluster' of pigment molecules plays an important role. Further binding of pigments to the 'peripheral binding sites' appeared also to stabilize the protein secondary structure of the reconstituted complexes.

The mode of action of glufosinate in algae: The role of uptake and nitrogen assimilation pathways

AbstractThe purpose of this work was to investigate the in‐vivo mode of action of glufosinate in comparison with that of the known glutamine synthetase inhibitor, methionine sulfoxmine, in photoautotrophic microorganisms. Using the eukaryotic green alga Chlorella fusca (Shih. & Krauss) and the cyanobacterium Anacystis nidulans (Kratz & Myers), currently developed [15N]NMR pulselabelling techniques have been employed to study the inhibition of ammonia assimilation. The results show that, while methionine sulfoximine immediately blocks glutamine synthesis, glufosinate action requires an induction process in both organisms investigated, possibly indicating the de‐novo synthesis of an amino acid membrane carrier. In addition, the observed ability of C. fusca to incorporate nitrogen into glutamate under glutamine synthetase‐inhibiting conditions is explicable by an anabolic function of a glutamate dehydrogenase in this organism. This might explain the large differences in observed species sensitivity to glufosinate exposure.

In vivo O-dealkylation of resorufin and coumarin ethers by the green alga Chlorella fusca analysed by a rapid and sensitive high-performance liquid chromatographic assay

The O-dealkylation of resorufin or coumarin ethers is known to be a cytochrome P450-mediated reaction. It is used as a reference to characterize the corresponding enzyme activity and its state of induceability. In adopting this approach, an in vivo O-dealkylase assay was developed (a) using the unicellular green alga Chlorella as the biotransforming system and (b) applying a rapid and sensitive high-performance liquid chromatography (HPLC) assay which makes use of the wide pH stability of modified aluminium oxide packings. Separation was achieved isocratically at pH 9.8 using an Aluspher RP select B analytical column. The eluate was monitored by fluorescence detection with a detection limit of ⩽3.5 pg for both resorufin and umbelliferone. The time needed for HPLC analysis is about 2 min, indicating that the method is suitable for automation.

Isolation and characterization of two different flavodoxins from the eukaryote Chlorella fusca.

Two different molecular forms of flavodoxin from the green alga Chlorella fusca have been purified to homogeneity and their properties compared. The molecular masses are 22 kDa (flavodoxin I) and 20 kDa (flavodoxin II). Western blots of axenic crude extract show the two bands. Both are single polypeptide chains and their N-terminal sequences differ but are very similar. Each form contains 1 mol of FMN/mol of apoprotein, exhibits a typical flavodoxin u.v.-visible absorption spectrum and does not contain covalently bound phosphate. The oxidation-reduction properties of the FMN in the flavodoxins differ considerably. Redox potentials of flavodoxin I at pH8 are -240 mV for the oxidized/semiquinone couple and -350 mV for the semiquinone/hydroquinone couple. Flavodoxin II gives more electronegative values: -278 mV and -458 mV respectively. Flavodoxin II fulfils better the redox requirements for photosynthetic electron transport and, as expected, it is more efficient at mediating NADP+ photoreduction in the photosynthetic electron flow. A new h.p.l.c. method for flavodoxin purification is described, which is useful for the isolation of very similar anionic proteins.

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Reconstitution of Light-Harvesting Complexes from Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae)

Abstract Reconstitution experiments of light-harvesting complexes were performed with the green alga Chlorella fusca and the chlorophyll c-containing prasinophyte Mantoniella squamata using a modified method according to Plumley and Schmidt [Proc. N atl. Acad. Sei. U .S.A . 84, 146 -150 (1987)]. Changing the pigment supply quantitatively or qualitatively in the reconsti­tution mixture homologous and heterologous reconstitutes were obtained. In contrast to higher plants, light-harvesting polypeptides from green algae are able to bind the chlorophylls as well as the xanthophylls in different stoichiometries. Heterologous reconstitutes of M . squamata polypeptides give further evidence for a rather high flexibility of pigment recog­nition and binding. This is the first report of successful reconstitution of a chlorophyll c-binding protein. Contrary to chlorophyll c-less light-harvesting complexes, the reconstitution of M . squamata is strongly pH-controlled. In summary, the results give evidence for a high specificity of porphyrin ring recognition and variability in xanthophyll binding capacity. Therefore, it is suggested that at least in algal light-harvesting proteins chlorophyll organiza­tion may be determined by other mechanisms than xanthophyll binding.

Studies on surfactant toxicity to the freshwater alga Chlorella fusca: a common mode of action?

In order to study whether the impact of surfactants on algae depends on a common mode of action, the eukaryotic green alga Chlorella fusca was subjected to two surfactants with identical lipophilic chains (C 16), the nonionic diethyleneglycol monohexadecylether (C16E2) and the cationic cetyltrimethylammoniumchloride (CTAC). Concentration-effect relationships for growth (cell volume) and reproduction (cell number) of synchronized C. fusca were determined for a surfactant exposure of 24 h. The observed-effect concentrations for both surfactants range from 10 −7 to 5·10 −6 molar. Exposure to the C16E2 surfactant resulted in a concomittant inhibition of both cell growth and reproduction. The CTAC surfactant, in contrast, induced a strikingly different concentration-response relationship for both parameters. To find the reason for the observed inhibition, photosynthesis and respiration of C fusca were determined during acute (30 min) and after prolonged (24 h) exposure to the surfactants. Only the cationic CTAC surfactant induced a moderate acute inhibition of photosynthetic oxygen production of C. fusca, which is constant even after prolonged exposure. C16E2, in contrast, failed to inhibit photosynthesis during short-term exposure, while prolonged incubation resulted in a distinct inhibition. The alga's respiration was only slightly affected during acute exposure to CTAC, while prolonged exposure induced a considerable effect. The nonionic C16E2, on the other hand, immediately inhibits the respiration of C. fusca, resulting in an almost total inhibition of respiration after long-term exposure. The results suggest that surfactant toxicity to algae can be attributed to an interaction with membrane functions of photosynthesis and mainly respiration. In addition, however, the cationic CTAC surfactant is shown to interfere especially with cell reproduction. More than one mode of action of cationic surfactants on algae, therefore, has to be assumed.

Herbicide bioconcentration in algae: studies on lipophilicity-sorption-activity relationships (LSAR) with Chlorella fusca

Lipophilicity-sorption-activity relationships (LSAR) were studied for a congeneric series of 15 herbicidal phenylureas with the unicellular green alga Chlorella fusca as a model organism. Data for substance lipophilicity (log k w), algal bioconcentration (log BCF) and algal toxicity (p I 50 CV/CN) were experimentally determined and subsequently subjected to a series of regression analyses. These studies revealed only moderate correlations between log k w and log BCF and between log k w and p I 50 values. In contrast, a highly significant relationship was observed between bioconcentration and toxicity. The following conclusions were drawn on basis of the results. (i) The predictive value of lipophilicity for estimating herbicide bioconcentration and toxicity in microalgae is limited. (ii) Different levels of phenylurea toxicity for Chlorella seem to be attributable to different degrees of bioconcentration. (iii) An integrated consideration of lipophilicity, bioconcentration and toxicity should be advantageous in aquatic ecotoxicological hazard assessment.

In Situ Nuclear Magnetic Resonance of 15N Pulse Labels Monitors Different Routes for Nitrogen Assimilation

Nuclear magnetic resonance offers the possibility of noninvasive in situ observation of (15)N pulse labeling in the presence of light. In vivo, exclusively the delta-nitrogen of Gln is labeled in the cyanobacterium Microcystis firma when glutamate synthase is inhibited by azaserine. In contrast, the green alga Chlorella fusca is additionally capable of incorporating nitrogen into Glu, thus providing evidence for an anabolic function of glutamate dehydrogenase in this organism.