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Ion Absorption and Retention by Chlorella Pyrenoidosa. II. Permeability of the Cell to Sodium and Rubidium.

The Na and Rb permeability of Chlorella pyrenoidosa were estimated from the rates of radioisotope self-diffusion.The isotopic exchange in absence of net ionic movements followed first order kinetics. This suggested that for sodium, which reached isotopic equilibrium in approximately 90 minutes, the cell behaved as 1 compartment with respect to isotopic exchange. Rubidium in 180 minutes approached isotopic equilibrium by 67%; thus, the existence of a single compartment for Rb has not been demonstrated. Net fluxes, calculated from the isotope exchange data, and expressed on a dry weight and surface area base showed that Na fluxes were approximately 7 times larger than Rb fluxes. Net Na fluxes of 90 milli-equivalents per 100 g dry weight per hour were far in excess of the observed maximum net accumulation of Na. However, Rb fluxes of 13 milliequivalents per 100 g dry weight per hour were of similar magnitude as the rate of Rb accumulation. Thus, permeability could be a limiting factor for Rb but not for Na accumulation. Sodium and Rb fluxes in absence of net ionic movements were inhibited by low temperature, dark air and dark N(2) conditions. This change in flux rates was explained mainly on the basis of metabolically dependent changes in the cell surface layers.Isotope fluxes of Rb were drastically reduced in dark air and dark N(2) in the absence or presence of net cation movements. Dark N(2) essentially eliminated net cation accumulation, whereas dark air had relatively little effect on the net K and Rb accumulation by Chlorella. Thus the 2 major factors involved in net cation accumulation in the Chlorella cell, permeability and processes leading to cation retention, respond differently to metabolic inhibition permitting a separation of these 2 important aspects of cation accumulation.

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Studies on the Biosynthesis and Metabolism of delta-Aminolevulinic Acid in Chlorella.

The regulation of chlorophyll synthesis in Chlorella was examined at the level of the formation and metabolism of delta-aminolevulinic acid. delta-Aminolevulinic acid synthetase activity could not be detected in broken cell preparations, and exogenously supplied delta-aminolevulinic acid was taken up only in the presence of dimethylsulfoxide, with a corresponding production of porphobilinogen.The delta-aminolevulinic acid dehydratase of Chlorella has a pH optimum of 7.8 and at this pH the Michaelis constant for delta-aminolevulinic acid is 0.13 millimolar.delta-Aminolevulinic acid excretion in the presence of levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, allowed measurement of the relative rates of delta-aminolevulinic acid synthesis under various growth conditions. A mutant which requires light for chlorophyll synthesis also requires light for delta-aminolevulinic acid accumulation in the presence of levulinic acid. This same mutant has 40% of the delta-aminolevulinic acid dehydratase activity of the wild-type Chlorella during growth in the dark on glucose.The necessity for protein synthesis during chlorophyll synthesis is due primarily to the requirement for protein synthesis during delta-aminolevulinic acid formation.It is concluded that the rate of chlorophyll formation and the cellular chlorophyll content are regulated by the relative rates of synthesis and breakdown of an enzyme responsible for delta-aminolevulinic acid biosynthesis and that this enzyme has an in vivo lifetime of about 30 minutes.

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A study of the control of glycolate excretion in chlorella.

Chlorella pyrenoidosa cells grown on 5% CO(2) excreted glycolate when incubated in light with 10 mm bicarbonate, but excreted no glycolate under the same conditions when they were maintained on air for 7 hours prior to the assay. Incubation of 5% CO(2)-grown and air-grown cells with 10 mm isonicotinyl hydrazide or 10 mm alpha-hydroxypyridinemethane sulfonate during the assay stimulated the excretion of glycolate by CO(2)-grown cells severalfold that of air-grown cells.Adaptation of CO(2)-grown Chlorella to growth on air did not affect the levels of glycolate dehydrogenase in the cells and did not affect the rate of dark oxidation and metabolism of exogeneous (14)C-glycolate by the cells. These results indicate that the lack of glycolate excretion by air-grown or air-adapted cells of Chlorella cannot be explained by a concomitant change in the level of glycolate dehydrogenase.

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Water permeability of chlorella cell membranes by nuclear magnetic resonance: measured diffusion coefficients and relaxation times.

Measurement by two nuclear magnetic resonance (NMR) techniques of the mean residence time tau(a) of water molecules inside Chlorella vulgaris (Beijerinck) var. "viridis" (Chodot) is reported. The first is the Conlon and Outhred (1972 Biochim Biophys Acta 288: 354-361) technique in which extracellular water is doped with paramagnetic Mn(2+) ions. Some complications in application of this technique are identified as being caused by the affinity of Chlorella cell walls for Mn(2+) ions which shortens the NMR relaxation times of intra- and extracellular water. The second is based upon observations of effects of diffusion on the spin echo of intra- and extracellular water. Echo attenuation of intracellular water is distinguished from that of extracellular water by the extent to which diffusive motion is restricted. Intracellular water, being restricted to the cell volume, suffers less echo attenuation. From the dependence of echo amplitude upon gradient strength at several values of echo time, the mean residence time of intracellular water can be determined. From the mean residence time of intracellular water, the diffusional water permeability coefficient of the Chlorella membrane is calculated to be 2.1 +/- 0.4 x 10(-3) cm sec(-1).

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Functional Comparison of the Photosystem II Center-Antenna Complex of a Phycocyanin-less Mutant of Cyanidium caldarium with That of Chlorella pyrenoidosa.

The photosystem II (PSII) antenna of a phycocyanin-less mutant (III-C) of Cyanidium caldarium was studied by means of O(2) activation and fluorescence induction measurements and compared to that of the green alga Chlorella pyrenoidosa.In Cyanidium this antenna contains only about 40 (Chl) a molecules per center yet shows PSII energy transfer as efficient as in Chlorella with 240 Chl a + b per center. In Cyanidium, PSII energy transfer occurs among at least four centers which are located close to each other. Cyanidium and Chlorella show the same PSII action and low temperature emission spectra, in the Chl a region despite the gross differences in antenna size.It is proposed that the PSII center-antenna complex found in Cyanidium is a core unit of the center-antenna complex of green algae; the only major difference between them is the addition of light-harvesting Chl a/b in the latter. It is further proposed that this core is the site of energy transfer between PSII centers.

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Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems.

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.

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Characterization of Nitrate Reductases from Corn Leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris: Sensitivity to a Proteinase Extracted from Corn Roots.

The sensitivity of the two forms of nitrate reductase, NR(I) and NR(II), obtained from the primary leaf of corn, to a limited action corn root proteinase has been examined. The corn inactivating protein (CIP) inhibited the overall reaction (NADH-NR) and the two partial reactions, cytochrome c reductase and reduced methyl viologen NR (MV-NR) of both forms of NR. NADH-cytochrome c reductase was more sensitive to the protease than MV-NR. NR(II) was less sensitive to inactivation than NR(I). When NR(I) and NR(II) were inactivated and then subjected to native gel electrophoresis the protein bands associated with MV-NR activity shifted from an R(m) value of 0.32 to 0.61 for NR(I) and from an R(m) of 0.28 to 0.60 for NR(II). For Chlorella NR these values are 0.32 and 0.70. The initial cleavage of the 116 kilodalton subunit of NR(I) yielded fragments of 84 and 80 kilodaltons after a 5 minute incubation with CIP. With longer incubation times smaller fragments were also identified. For the Chlorella NR the initial cleavage products are approximately 68 and 25 kilodaltons. Longer incubation times also led to smaller fragments. The products of hydrolysis by this limited action protease are quite different for the corn and Chlorella NRs.

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Metabolism of Fatty Acid Hydroperoxides by Chlorella pyrenoidosa.

The green alga Chlorella pyrenoidosa was examined for its ability to metabolize 13-hydroperoxylinoleic and 13-hydroperoxylinolenic acids. The study showed that Chlorella extracts possessed hydroperoxide dehydrase and other enzymes of the jasmonic acid pathway. However, under normal laboratory conditions for culture growth, neither jasmonic acid nor metabolites of the jasmonic acid pathway were present in Chlorella. In vitro enzyme studies also revealed the presence of hydroperoxide lyase activity that cleaved 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acid into two products, 13-oxo-cis-9,trans-11-tridecadienoic acid and pentane (from linoleic acid) or pentene (from linolenic acid). The lyase was heat-labile, insensitive to 50 millimolar KCN, and had an approximate molecular weight of 48,000 as estimated by gel filtration. Two other products, 13-hydroxy-cis-9,trans-11,cis-15-octadecatrienoic acid and 12, 13-trans-epoxy-9-oxo-trans-10,cis-15-octadecadienoic acid, were also observed. Because these compounds are also products of nonenzymic, Fe(II)-catalyzed hydroperoxide decomposition reactions, their presence suggested that the observed lyase activity may occur via a homolytic decomposition mechanism.

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Interactions between Glucose and Inorganic Carbon Metabolism in Chlorella vulgaris Strain UAM 101.

Chlorella vulgaris strain UAM 101 has been isolated from the effluent of a sugar refinery. This alga requires glucose to achieve maximal growth rate even under light saturating conditions. The growth rate of cultures grown on light + CO(2) + glucose (3.16 per day) reaches the sum of those grown on light + CO(2) (1.95 per day) and on dark + glucose (1.20 per day). Unlike other Chlorella strains, uptake of glucose (about 2 micromoles per milligram dry weight per hour) was induced to the same extent in the light and dark and was not photosensitive. The rate of dark respiration was not affected by light and was strongly stimulated by the presence of glucose (up to about 40% in 4 hours). The rate of photosynthetic O(2) evolution was measured as a function of the CO(2) concentration. These experiments were conducted with cells which experienced different concentrations of CO(2) or glucose during growth. The maximal photosynthetic rate was inhibited severely by growing the cells in the presence of glucose. A rather small difference in the apparent photosynthetic affinity for extracellular inorganic carbon (from 10-30 micromolar) was found between cells grown under low and high CO(2). Growth with glucose induced a reduction in the apparent affinity (45 micromolar) even though cells had not been provided with CO(2). Experiments performed at different pH values indicate CO(2) as the major carbon species taken from the medium by Chlorella vulgaris UAM 101.

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A study of the growth for the microalga Chlorella vulgaris by photo-bio-calorimetry and other on-line and off-line techniques.

Calorimetry and other on-line techniques are used for the first time as complement to the traditional off-line methods in order to follow the growth of the green Chlorella vulgaris microalgae. A 2-L photo-bio-reactor was adapted from a commercial calorimeter used previously to study heterotrophic microbial growth. An external source of light was added to favor the photosynthesis of the autotrophic cells. Heterotrophic growth was also tested with external glucose in the broth. A third mode, mixotrophic, allowed faster autotrophic plus heterotrophic growth. Calorimetric measurements were performed considering the corresponding calibrations in order to consider only the energy involved during the microalgal growth. The three different modes of Chlorella cultures were energetically characterized. Besides calorimetry, the weight of diluted nitric acid added to maintain the pH of the culture was correlated with the cellular growth and the nitrogen composition of the algae. Additionally, the on-line infrared spectroscopy proved to be an efficient technique to follow the composition of the broth in glucose, nitrates, and phosphates. These results were compared and complemented with some classic off-line techniques used to track this kind of cultures.

Biomass↗

A hot water extract of Chlorella pyrenoidosa reduces body weight and serum lipids in ovariectomized rats.

The effects of a hot water extract of Chlorella pyrenoidosa, which contains chlorella growth factor (CGF), on the body weight, serum lipids, and the bone mass were evaluated using an ovariectomized rat as a model for postmenopausal bone loss. Rats were divided into four groups: sham-operated (Sham), Sham given the CGF solution, ovariectomized (OVX), and OVX given the CGF solution, respectively. Administration of the extract to OVX rats suppressed the body weight gain. After 7 weeks, the administration of the extract to the OVX group reduced increases in both serum total cholesterols and high-density lipoprotein (HDL) cholesterols. It also normalized the decrease of triglyceride level in the OVX group. The ovariectomy decreased the tibial bone mineral density (BMD) by 19%, and the administration of the extract to OVX rats did not inhibit this decrease. These results suggest that a dietary supplement of CGF may be useful to control the body weight and improve lipid metabolism of menopausal women.

Animals↗

Effect of methyl parathion on the population growth of the rotifer Brachionus patulus (O. F. Müller) under different algal food (Chlorella vulgaris) densities.

The population growth of the rotifer Brachionus patulus was studied under a combination of different concentrations of Chlorella vulgaris and methyl parathion. To obtain sublethal concentrations of the pesticide a 24-h LC(50) bioassay was performed under two food densities (1.5 and 3.0x10(6) cells ml(-1)) using neonate rotifers. For the population growth experiments, seven toxicant concentrations were used ranging from 0.16 to 10.0 mg L(-1) and four food densities (0.75, 1.5, 3.0, and 6.0x10(6) cells ml(-1)), each with four replicates and thus in all, 128 test jars. A medium with Chlorella alone and without toxicant acted as control. The initial density of the rotifers in each replicate were 5 individual ml(-1). Experiments were conducted for 20 days. The LC(50) values of B. patulus were 8.8 and 10.7 mg L(-1) at low and high algal food density, respectively. Data on the population growth studies revealed a significant effect of both food density and toxicant concentration on the maximum abundance, day at which maximum abundance was reached, and rate of population increase per day. At any toxicant concentration, an increase in food density reduced the toxicity of methyl parathion to rotifers. In controls the maximum rotifer density under the highest food level (6x10(6) cells ml(-1)) was 795+/-46 individuals ml(-1). The rate of population increase per day (r) in controls varied from 0.22+/-0.01 to 0.34+/-0.01 depending on food density. Algal food density had a significant influence in reducing the toxicity of methyl parathion to B. patulus under both acute and chronic toxicity test conditions.

Animals↗

Expression of the gene encoding a translational elongation factor 3 homolog of Chlorella virus CVK2.

A gene encoding a putative translational elongation factor 3 (EF-3) on the genome of Chlorella virus CVK2 has been cloned and sequenced. It encodes for a predicted polypeptide of 1120 amino acids (aa) with a molecular mass of 127 kDa. The overall amino acid sequence of CVK2 EF-3 (vEF-3) showed 36.1% identity and 83.6% similarity to that of Saccharomyces cerevisiae EF-3. Functional domains including two sets of ATP-binding motifs were extremely well conserved between vEF-3 and yeast EF-3; 63.6% identity and 92.4% similarity in total 330-aa portions. Northern blot analysis indicated that the vEF-3 gene was transcribed in the host cells early, at 20 min postinfection (p.i.), as well as late, 3-4 hr p.i. Western blot analyses with anti-vEF-3 antibody detected the 120 kDa vEF-3 protein product after 40 min p.i. It was present until the final stages of infection but absent in the virion. The vEF-3 gene was highly conserved among all Chlorella viruses isolated in Japan.

Amino Acid Sequence↗

Alternative expression of a chitosanase gene produces two different proteins in cells infected with Chlorella virus CVK2.

Several Chlorella virus CVK2 proteins had chitosanase and/or chitinase activities. A gene coding for an ORF of 328 amino acids (aa) with a predicted molecular mass of 36,769 Da was cloned from the viral genome. The predicted amino acid sequence of an N'-portion (174 aa) of this gene product (vChta-1) showed 22 to 25% identity with various bacterial chitosanases. A glutathione S-transferase (GST)-vChta-1 fusion protein had strong chitosanase activity. Western blot analysis with antisera raised against the vChta-1 protein identified two proteins of 37 and 65 kDa in virus-infected Chlorella cells beginning at 240 min postinfection and continuing until cell lysis. The larger protein was packaged in the virion, while the smaller one remained in the cell lysate. Both chitosanase proteins were produced from the single gene, vChta-1, by a mechanism of alternative gene expression.

Alternative Splicing↗

Chlorella virus NY-2A encodes at least 12 DNA endonuclease/methyltransferase genes.

The 380-kb chlorella virus NY-2A genome is highly methylated; 45% of the cytosines are 5-methylcytosine (5mC) and 37% of the adenines are N6-methyladenine (6mA). Based on the sensitivity/resistance of NY-2A DNA to 80 methylation-sensitive DNA restriction endonucleases, the virus is predicted to encode at least 10 DNA methyltransferases: 7 6mA-specific methyltransferases, M.CviQI (GTmAC), M.CvQII (RmAR), M.CviQIII (TCGmA), M.CviQIV (GmATC), M.CviQV (TGCmA), M.CviQVI (GmANTC), and M.CviQVII (CmATG): and 3 5mC-specific methyltransferases, M.CviQVIII [RGmC(T/C/G)], M.CviQIX (mCC), and M.CviQX (mCGR). Five of the 6mA methyltransferase genes, M.CviQI, M.CviQIII, M.CviQV, M.CviQVI, and M.CviQVII, were cloned and sequenced. In addition, 2 site-specific endonuclease activities, R.CviQI (G/TAC) and NY2A-nickase (R/AG), were detected in cell-free extracts from NY-2A virus-infected chlorella. Therefore, the NY-2A genome contains at least 12 DNA methyltransferase and endonuclease genes which, altogether, compose about 3-4% of the virus genome.

Amino Acid Sequence↗

Chlorella virus PBCV-1 encodes functional glutamine: fructose-6-phosphate amidotransferase and UDP-glucose dehydrogenase enzymes.

DNA sequence analysis of the 330-kb Chlorella virus PBCV-1 genome unexpectedly revealed several open reading frames which encode proteins that are homologous to sugar-manipulating enzymes including glutamine:fructose-6-phosphate amidotransferase (GFAT), UDP-glucose dehydrogenase (UDP-GlcDH), and hyaluronan synthase (HAS). PBCV-1 genes encoding the putative GFAT and UDP-GlcDH enzymes were expressed in Escherichia coli, and both recombinant proteins have the predicted enzyme activity in cell free extracts. These same two genes are transcribed early in PBCV-1 infection, and both genes are widespread among the Chlorella viruses. The products of the reactions catalyzed by these two enzymes are precursors in the biosynthesis of hyaluronan polysaccharide. Previous experiments established that, like the GFAT and UDP-GlcDH genes, the HAS gene is transcribed early and encodes a functional enzyme (DeAngelis, P. L., Jing. W., Graves, M. V., Burbank, D. E., and Van Etten, J. L. (1997) Science 278, 1800-1803). Interestingly, the predicted amino-acid sequences of the PBCV-1 GFAT and UDP-GlcDH enzymes are more similar to bacterial GFAT and UDP-GlcDH enzymes than to their eukaryotic counterparts. In contrast, the amino-acid sequence of the PBCV-1 HAS enzyme more closely resembles eukaryotic enzymes.

Animals↗

Genetic variation of chlorella viruses: variable regions localized on the CVK2 genomic DNA.

A physical map of the Chlorella virus CVK2 genomic DNA has been constructed based on a cosmid contig covering the entire genomic region. By using Southern blot analysis with 22 gene probes, the gene arrangement along the genome was compared between CVK2 and PBCV-1, the prototypic member of Phycodnaviridae, whose genomic sequence is now available. The major rearrangements were (1) an insertion of a 20-kbp region around the left end of CVK2 DNA, (2) a duplication of the gene for major capsid protein in CVK2 DNA, (3) deletions/insertions of some open reading frames, and (4) divergence in the terminal inverted repeat sequences. Despite these changes, extensive colinearity was revealed between most of the genes along the CVK2 and PBCV-1 genomes. These data imply that the Chlorella virus genome has an overall high degree of genomic stability, encompassing specific islands of rearrangements.

Amino Acid Sequence↗

Expression of a chitinase gene and lysis of the host cell wall during Chlorella virus CVK2 infection.

A chitinase gene (vChti-1) encoded by the Chlorella virus CVK2 was cloned and characterized. The vChti-1 open reading frame consisted of 2508 bp corresponding to 836 amino acid residues. The predicted amino acid sequence contained two sets of a family 18 catalytic domain that is responsible for chitinase activity. Northern blot analysis revealed that the vChti-1 gene was expressed in virus-infected Chlorella cells late in infection, when a single transcript of about 2.5 kb appeared at 120 min postinfection. This result was confirmed by Western blotting with a specific anti-vChti-1 protein antibody; a protein of about 94 kDa was detected specifically beginning at 240 min postinfection and was present until cell lysis. The protein was not incorporated into viral particles but remained in the medium after cell lysis. The vChti-1 protein produced in virus-infected cells showed chitinase activity on zymogram assays.

Amino Acid Sequence↗