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PubMed2000

Mapping of cDNA clones on contig of Chlorella chromosome I.

Complementary DNA (cDNA) clones specific to the smallest chromosome (chromosome I) of Chlorella vulgaris C-169 were selected from cDNA libraries with probes of chromosome I DNA fragments amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). A total of 15 clones was obtained, which included gene homologs for alpha-tubulin, inosine-5'-monophosphate dehydrogenase, beta-1,4-mannase, a TTG-binding protein, a heat shock protein, thioredoxin/protein disulfide isomerase, transcription factor NF-E2, an oxidoreductase, and UDP-n-acetylglucosamine enolpyruvyltransferase. These clones were definitely localized at specific sites on the chromosome I physical map constructed on the basis of overlapping cosmid clones (the contig). They were predominantly distributed within the left two-thirds of the chromosome. This contrasts with the distribution of repetitive elements such as short interspersed elements (SINEs), which are rather abundant in the right two-thirds of chromosome I. The comparative simplicity of the gene arrangement of Chlorella chromosome I suggests that it may be able to serve as a prototypic system for deciphering the complexity of huge plant chromosomes.

Journal Article↗
PubMed2004

Effect of partial shading on photoproduction of hydrogen by Chlorella.

The photoproduction of hydrogen by the green alga Chlorella pyrenoidosa was studied in 650-ml bubble columns made of glass with 7 cm diameter. Hydrogen was produced by adding sodium dithionite as an oxygen scavenger directly to an algal suspension. When a part of the wall of the bubble column was shaded with a sheet of plastic film impervious to light, the production rate and total volume of hydrogen increased as compared to those in the columns without partial shading. This relationship between the hydrogen photoevolution rate and the proportion of lighted region is contrary to normal photochemical reactions. This phenomenon is considered to be related to the regulation by light of the activity of the enzymes and/or the photosynthetic electron transport systems, which was examined by measuring fluorescence induction curves of dark-adapted Chlorella cells and also the distribution of light intensity within the bubble column bioreactors.

Journal Article↗
PubMed2004

Production of intracellular phytochemicals in Chlorella under heterotrophic conditions.

A heterotrophic synchronous culture (HSC) of Chlorella regularis S-50, a strain with a high growth rate, and one for its mutant were established by alternately culturing for 6 h on medium containing glucose and for 3 h on medium without glucose. The changes in cellular components and respiratory activity during the course of cell cycling in the HSC were investigated. The synchronized daughter cells (small cells) produced by the HSC contained 2-3 times as much intracellular phytochemicals (carotenoids, chlorophyll, tocopherols, and others typical of green plants) as the glucose-metabolizing cells or the non-synchronous cell mass. To attain the HSC at high cell density, the effects of glucose and oxygen on growth rate, synchronous growth, and production of intracellular phytochemicals were revealed. During the increase in cell mass, when a glucose concentration of 0.5-10 g l(-1) in the culture fluid was maintained by glucose feeding, and the (Qo2)(max) in cells was kept constant by supplying oxygen, cell mass increased synchronously with a specific growth rate, mu, of over 0.2 h(-1). In the HSC, when the late glucose-metabolizing cells were aerated by supplying oxygen to maintain over a half of the (Qo2)(max) under glucose-deficient conditions, intracellular phytochemicals increased rapidly in parallel with cell division. On the basis of these results, the HSC system at high cell density was attained by a glucose-limited fed-batch culture that involves three controlled conditions; glucose concentration, glucose feeding time, and oxygen supply. The system maintained synchronous growth for more than three generations until the cell density reached about 90 g l(-1) with a mu of 0.2 h(-1). In the HSC system, synchronized daughter cells were obtained at a cell productivity of 84 g l(-1) (30 h)(-1). The cell yield for glucose was 0.45. A weight of 100 g of dry cells contained 710 mg carotenoids, 350 mg lutein, 50 mg alpha-carotene, 60 mg beta-carotene, 3.3 g chlorophylls, 23 mg tocopherols, and others, with 63 g of protein rich in essential amino acids. Industrial-scale HSC systems made it possible to steadily produce Chlorella, containing 10-50 times as much phytochemicals as green and yellow vegetables, regardless of the weather.

Journal Article↗
PubMed2001

Non-photochemical chlorophyll fluorescence quenching and structural rearrangements induced by low pH in intact cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae).

We have used circular dichroism (CD) spectroscopy and chlorophyll fluorescence induction measurements in order to examine low-pH-induced changes in the chiral macro-organization of the chromophores and in the efficiency of non-photochemical quenching of the chlorophyll a fluorescence (NPQ) in intact, dark-adapted cells of Chlorella fusca (Chlorophyceae) and Mantoniella squamata (Prasinophyceae). We found that: (i) high proton concentrations enhanced the formation of chiral macrodomains of the complexes, i.e. the formation of large aggregates with long-range chiral order of pigment dipoles; this was largely independent of the low-pH-induced accumulation of de-epoxidized xanthophylls; (ii) lowering the pH led to NPQ; however, efficient energy dissipation, in the absence of excess light, could only be achieved if a substantial part of violaxanthin was converted to zeaxanthin and antheraxanthin in Chlorella and Mantoniella, respectively; (iii) the low-pH-induced changes in the chiral macro-organization of pigments were fully reversed by titrating the cells to neutral pH; (iv) at neutral pH, the presence of antheraxanthin or zeaxanthin did not bring about a sizeable NPQ. Hence, low-pH-induced NPQ in dark adapted algal cells appears to be associated both with the presence of de-epoxidized xanthophylls and structural changes in the chiral macrodomains. It is proposed that the macrodomains, by providing a suitable structure for long-distance migration of the excitation energy, in the presence of quenchers associated with de-epoxidized xanthophylls, facilitate significantly the dissipation of unused excitation energy.

Journal Article↗
PubMed2002

Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants.

In non-photosynthetic, yellow or colourless mutant cells of Chlorella kessleri, grown with nitrate as sole nitrogen source, blue light inhibited the uptake of the amino acids glycine, proline and arginine and of ammonia in growing cells, while it enhanced the uptake of these amino acids in resting cells. On the other hand, in cells grown with ammonia as the only nitrogen source without nitrate reductase activity, blue light did not influence the uptake of amino acids and of ammonia in growing cells, while it enhanced the uptake of amino acids in resting cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to growing cells, resulted in intracellular ammonia-accumulation and inhibition of uptake of glycine and of ammonia. For the colourless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of ammonia seems to be influenced by nitrate reductase and the uptake of amino acids was influenced by both nitrate reductase and an unknown blue-light-receptor(s). The uptake of urea in mutant cells is not influenced by the irradiation with blue light. Uptake of glycine was also increased after addition of glucose (hexose) in the dark. Because blue light is known to enhance the breakdown of starch, a reaction producing glucose for oxidative degradation in the algae used, the role of glucose (hexose) in the blue light-affected uptake of amino acids is discussed.

Journal Article↗
PubMed1982

Viruses of symbiotic Chlorella-like algae isolated from Paramecium bursaria and Hydra viridis.

We previously reported that isolation of symbiotic Chlorella-like algae from the Florida strain of Hydra viridis induced replication of a virus (designated HVCV-1) in the algae. We now report that isolation of symbiotic Chlorella-like algae from four other sources of green hydra and one source of the protozoan Paramecium bursaria also induced virus synthesis. Algae from one of these hydra contained a virus identical to HVCV-1 (based on its rate of sedimentation, buoyant density, reaction to HVCV-1 antiserum, and DNA restriction fragments) whereas algae from the other three hydra contained another similar, but distinct, virus (designated HVCV-2). The virus from the paramecium algae (designated PBCV-1) was distinct from both HVCV-1 and HVCV-2. The symbiotic algae in the hydra could also be distinguished ultrastructurally. Chloroplasts of both algae that produced HVCV-1 lacked a pyrenoid whereas chloroplasts of the other three symbiotic algae contained pyrenoids. Since all symbiotic eukaryotic algae we have examined have had virus, a potential viral role in symbiosis is suggested.

Journal Article↗
PubMed1986

Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA.

The organization of a cloned rRNA gene cluster from Chlorella ellipsoidea chloroplast DNA (cpDNA) has been analyzed. Southern hybridization experiments with labelled chloroplast rRNAs as probes revealed an extraordinarily large size of the 16S-23S rRNA spacer region, ca. 4.8 kbp, almost twice as large as those of most higher plants. The nucleotide sequence determined on this region has shown that: (1) The tRNAIle gene locating in this region is similar to those of higher plant chloroplasts, blue-green algae and E. coli but does not contain any introns in contrast to higher plant chloroplasts. (2) The tRNAAla gene is absent from this region. (3) There are four open reading frames (ORFs) coding for 55, 102, 107 and 110 amino acids, respectively. (4) A few sets of unique sequence were found repeatedly in this region. (5) The 23S rRNA gene is coded on the opposite strand in the reverse order. This arrangement of the 16S-23S rRNA region of Chlorella cpDNA is quite different from any of those reported so far for various organisms.

Chloroplasts↗
PubMed1998

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.

DNA↗
PubMed2004

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification.

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.

Amino Acid Sequence↗
PubMed1996

A New Screening Method for Algal Photosynthetic Mutants (CO2-Insensitive Mutants of the Green Alga Chlorella ellipsoidea).

A new method has been developed for screening algal photosynthetic mutants. This method uses autoradiography to assess gross photosynthetic 14C fixation by green algal colonies on agar plates and allows the identification of clones that differ in photosynthetic characteristics from wild-type cells. Three wild-type cells, high-CO2-grown Chlorella ellipsoidea, air-grown C. ellipsoidea, and air-grown Chlorella saccharophila, had K0.5 values for dissolved inorganic carbon (DIC) of 1083, 250, and 50 [mu]M, respectively, and as plaques on agar plates at Chl densities greater than 25 [mu]g cm-2 exhibited relative amounts of 14C fixation of 15, 55, and 100%, respectively. Cells of C. ellipsoidea were mutagenized with x-rays and screened by this method. Growth of C. ellipsoidea in high CO2 represses DIC transport and thus lowers its affinity for DIC. Five of the mutants detected by this method showed high-affinity photosynthesis similar to air-grown wild-type cells even when grown in high CO2. Seven other mutants when grown in high CO2 showed affinities for DIC intermediate between air-grown and high-CO2-grown wild-type cells. The affinities of high-CO2-grown mutants were reflected precisely in their capacities to accumulate DIC intracellularly. These results indicate that the mutants are fully or partially insensitive to the repressive effect of ambient CO2 concentration on DIC transport.

Journal Article↗
PubMed1966

Responses of Heterotrophic Cultures of Chlorella vulgaris Beyerinck to Darkness and Light. I. Pigment and pH Changes.

Glucose cultures of Chlorella vulgaris were grown in white light, in monochromatic light, and in darkness. Difference spectra showed that all wavelengths resulted in increased pigmentation over the dark controls.Cells irradiated with the 600 mmu beam showed a much higher absorption in the blue end of the spectrum with respect to the red end than is normally found in absorption spectra of white-light grown Chlorella cells.Dry weight comparisons between monochromatic light and dark controls showed the controls to be somewhat higher. This demonstrated that the monochromatic irradiation produced pigment synthesis but no increase in growth. Dark growth experiments suggested that cultures incubated in darkness on glucose excreted an acidic product.

Journal Article↗
PubMed1966

Correlation of alpha-Linolenate to Photosynthetic O(2) Production in Chlorella.

Photosynthetic oxygen evolution per milligram of chlorophyll in Chlorella vulgaris varies with the age of the culture. The rate of oxygen evolution is low in the starting cells, it rises to a maximum after 24 hours of growth and then declines to the initial low value after 72 to 90 hours. These changes in photosynthetic competence of chlorophyll in Chlorella are paralleled by changes in alpha-linolenate per milligram of chlorophyll. In general the magnitude of the photosynthetic competence of chlorophyll is directly proportional to the magnitude of the ratio of alpha-linolenate to chlorophyll, regardless of whether high ratios are due to high alpha-linolenates or low chlorophyll values. This relationship holds when the cultures are grown either under continuous or intermittent illumination.

Journal Article↗
PubMed1966

Hydrogenase mediated nitrite reduction in chlorella.

The assay of the hydrogenase of glucose-grown cells of Chlorella pyrenoidosa, strain 7-11-05 by means of nitrite reduction with molecular hydrogen is described. The hydrogenase of Chlorella shows maximum activity immediately after equilibration in the hydrogen atmosphere. The hydrogenase mediated reduction of nitrite to ammonia requires the presence of CO(2). However, at pH 6.4. when the reaction proceeds optimally, there is apparently sufficient retention of metabolic CO(2) to support the reaction, which goes to completion, at near maximum rates.Reduction of nitrite in the hydrogenase system when CO(2) is present results in the uptake of 3 moles of H(2) per mole of nitrite and ammonia is the product. When CO(2) is absent or limiting, ammonia is also formed from nitrite but with the uptake of less than the stoichiometric amount of H(2). It is concluded that CO(2) is essential for the uptake of H(2), and that in the absence of CO(2) internal hydrogen donors support nitrite reduction.The possibility that CO(2) exerts a catalytic effect in all reductions mediated by hydrogenase in algae is considered, and a further hypothesis, that hydrogenase arises from that portion of the photosynthetic machinery which also shows a catalytic requirement for CO(2), is proposed.

Journal Article↗
PubMed1966

Lipid synthesis in the presence of nitrogenous compounds in Chlorella pyrenoidosa.

Acetate was incorporated by Chlorella pyrenoidosa into nonvolatile water-soluble compounds (amino acids, organic acids and carbohydrates) with pH optimum between 4 and 5. Incorporation into lipid was maximal at pH 7.5. The proportion of incorporated acetate in lipid was not significantly affected by acetate concentration and chlorophyll concentration in the ranges tested. Illumination of Chlorella during acetate metabolism increased the synthesis of lipid with concomitant decrease in the synthesis of water-soluble compounds. Nitrate and ammonium ions had essentially no effect on acetate metabolism. Inhibition by nitrite was greater on the synthesis of lipid. Illumination reversed the gross inhibition by nitrite, but altered the distribution of incorporated label in favor of the water-soluble compounds.

Eukaryota↗
PubMed1967

Ion Absorption and Retention by Chlorella pyrenoidosa. III. Selective Accumulation of Rubidium, Potassium and Sodium.

The selective preference of Chlorella pyrenoidosa for alkali metal cations was found to have the order Rb > K > > > Na. It was demonstrated that a cation of higher preference can replace in the cell cations of lower preference by an ion interchange process.The replacement of Na from the cell by K or Rb occurred against high external Na concentrations up to 450 meq/1 Na.It is suggested that the structural selectivity of the Chlorella cell may be amplified by a chromatographic type repetitive selection process, driven by metabolically dependent unsymmetric shape changes of cellular membranes.

Journal Article↗
PubMed1968

Studies of Sulfate Utilization by Algae. 6. Adenosine-3'-Phosphate-5'-Phosphosulfate (PAPS) as an Intermediate in Thiosulfate Formation From Sulfate by Cell-Free Extracts of Chlorella.

When cell-free preparations of Chlorella pyrenoidosa Chick (Emerson strain 3) form thiosulfate from labeled sulfate, another radioactive compound also appears. This compound has been isolated in quantity and is shown to be identical with adenosine-3'-phosphate-5'-phosphosulfate (PAPS) on the basis of its chromatographic and electrophoretic behavior, chemical composition, sensitivity to selective degradative enzymes, and its ability to serve as a substrate for rat liver aryl sulphotransferase. In addition, as expected for PAPS, the compound on mild acid treatment yields all of its radioactive sulfur as sulfate, and is converted to a compound identical with adenosine-3',5'-diphosphate (PAP). Replacement of sulfate and ATP by this PAP(35)S in the usual incubation mixture yields the same product, thiosulfate, which can be isolated as such or detected as acid-volatile radioactivity. This conversion of PAP(35)S to thiosulfate still requires the addition of Mg(2+) and a reductant such as 2,3-dimercaptopropan-1-ol (BAL). The cause of our previous result that high concentrations of ATP inhibit thiosulfate formation from sulfate can be ascribed to a small amount of PAP contaminating the ATP preparations, since PAP proves to be an exceedingly effective inhibitor of the conversion of PAP(35)S to thiosulfate. Sulfate reduction to thiosulfate by Chlorella extracts is discussed and compared with similar systems from other organisms.

Journal Article↗
PubMed1968

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella.

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with (35)S in either the SH- or SO(3) - sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO(3)-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO(3)-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO(3)-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO(3)-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.

Journal Article↗
PubMed1968

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris.

An enzyme catalyzing the formation of delta-aminolevulinic acid by transamination of gamma,delta-dioxovaleric acid with l-alpha-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in delta-aminolevulinic acid metabolism in plants is not clearly understood.

Journal Article↗