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GROWTH OF CHLORELLA PYRENOIDOSA IN RECYCLED MEDIUM.

Bench-scale studies with Chlorella pyrenoidosa 7-11-05 were conducted in a 4-liter culture vessel with a used and recycled medium. Algal cultures were maintained for periods of several weeks by supplementing the nutrient medium with minimal amounts of certain salts. An algal strain was maintained for a period of up to 72 days with a supplemented recycled medium. No inhibition was observed as the result of any autotoxic materials. Rather dense cultures were maintained in the presence of high bacterial populations.

Chlorella↗

GAS EXCHANGE OF ALGAE. I. EFFECTS OF TIME, LIGHT INTENSITY, AND SPECTRAL-ENERGY DISTRIBUTION ON THE PHOTOSYNTHETIC QUOTIENT OF CHLORELLA PYRENOIDOSA.

Continuously growing cultures of Chlorella pyrenoidosa Starr 252, operating at constant density and under constant environmental conditions, produced uniform photosynthetic quotient (PQ = CO(2)/O(2)) and O(2) values during 6 months of observations. The PQ for the entire study was 0.90 +/- 0.024. The PQ remained constant over a threefold light-intensity change and a threefold change in O(2) production (0.90 +/- 0.019). At low light intensities, when the rate of respiration approached the rate of photosynthesis, the PQ became extremely variable. Six lamps of widely different spectral-energy distribution produced no significant change in the PQ (0.90 +/- 0.025). Oxygen production was directly related to the number of quanta available, irrespective of spectral-energy distribution. Such dependability in producing uniform PQ and O(2) values warrants a consideration of algae to maintain a constant gas environment for submarine or spaceship use.

Carbon Dioxide↗

Augmentation of host defense by a unicellular green alga, Chlorella vulgaris, to Escherichia coli infection.

Protection against Escherichia coli inoculated intraperitoneally into mice was enhanced by intraperitoneal, intravenous, or subcutaneous administration of a water-soluble, high-molecular-weight fraction extracted from a dialyzed hot-water extract from a strain of Chlorella vulgaris (CVE-A). The enhancing effect was detected with doses over 2.0 mg/kg and when doses were administered 1, 4, or 7 days before the infection. The elimination of bacteria from the spleen of CVE-A-treated mice was increased, and this enhanced elimination may have been related to the acceleration of superoxide generation and chemokinesis in polymorphonuclear leucocytes by CVE-A treatment. A cyclophosphamide-induced decrease in protection against E. coli could be prevented by subcutaneous administration of CVE-A.

Animals↗

Metabolic control of urea catabolism in Chlamydomonas reinhardi and Chlorella pyrenoidosa.

In the unicellular green alga Chlamydomonas reinhardi (strain y-1), synthesis of the enzymes required for urea hydrolysis is under substrate induction control by urea and under end product repression control by ammonia. Hydrolysis of urea if effected by the sequential action of the discrete enzymes urea carboxylase and allophanate lyase, collectively called urea amidolyase. The carboxylase converts urea to allophanate in a reaction requiring biotin, adenosine 5'-triphosphate, and Mg2+. The lyase hydrolzyes allophanate to ammonium ions and bicarbonate. Neither activity is present in more than trace amounts when cultures are grown with ammonia or urea plus ammonia, or when they are starved for nitrogen for 8 h. Urea in the absence of ammonia induces both activities 10 to 100 times the basal levels. Addition of ammonia to an induced culture causes complete cessation of carboxylase accumulation and an 80% depression of lyase accumulation. Ammonia does not reduce urea uptake by repressed cells, so it does not prevent induction by the mechanism of inducer exclusion. The unicellular green alga Chlorella pyrenoidosa (strain 3 Emerson) also has discrete carboxylase and lyase enzymes, but only the carboxylase exhibits metabolic control.

Ammonia↗

Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella.

Earlier studies indicated that the gene of an ammonium-inducible glutamate dehydrogenase (GDH) was inducible throughout the cell cycle and was expressible shortly after replication early in the S-phase in synchronous Chlorella cells growing at a rate of 13% per h in the absence of inducer. In the present study, synchronous cells cultured at the same growth rate in the continuous presence of inducer accumulated this enzyme in a linear manner, with a positive rate change observed late instead of early in the S-phase. At a growth rate of 26% per h, the positive rate change appeared to be displaced to 1.5 h before the S-phase in the next cell cycle. With 2'-deoxyadenosine, an in vivo inhibitor of deoxyribonucleic acid (DNA) synthesis, the magnitude of the positive rate change was shown to be proportional to the relative increase in DNA in the previous cell cycle. Collectively, these data support the idea that expression of newly replicated genes of this enzyme can be delayed into the subsequent cell cycle in cells in the continuous presence of inducer. Studies with cycloheximide indicated that the inducible GDH and another GDH isozyme were stable in fully induced cells in the absence of protein synthesis. However, after ammonium was removed from the culture medium, the activity of the inducible GDH decreased rapidly in vivo, with a half-time of 5 to 10 min at 38.5 degrees C, whereas the rate of accumulation of the other GDH isozyme did not change. Addition of cycloheximide, at the time of inducer removal, prevented this loss in activity of the inducible GDH. The inability to rescue the activity of the inducible GDH, by readdition of ammonium during the deinduction period, indicates that this enzyme probably underwent irreversible inactivation and/or proteolytic degradation.

Chlorella↗

Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana.

The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism.

Amino Acids↗

Regulation of initial rate of induction of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase during the cell cycle of synchronous Chlorella.

When synchronous cells of the eucaryotic microorganism Chlorella sorokiniana growing in nitrate medium were challenged to synthesize an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) at frequent intervals during the cell cycle the initial rate of induction (i.e., enzyme potential) of this enzyme increased in an approximately linear manner until the period of DNA replication (i.e., S phase). During the S phase, NADP-GDH potential exhibited a positive rate change proportional to the step increase in DNA level. The timing of this rate change was insensitive to large changes in cellular growth rate. This rate change could be blocked within the first cell cycle by specific inhibition of DNA replication with 2'-deoxyadenosine. The approximately linear increase in NADP-GDH potential and also of total cellular protein observed before and after the S phase is proposed to be a result of the increasing photosynthetic capacity of the cell during the cell cycle.

Cell Cycle↗

Turnover of ammonium-inducible glutamate dehydrogenase during induction and its rapid inactivation after removal of inducer from Chlorella sorokiniana cells.

When ammonia was removed from Chlorella sorokiniana cells, which contain an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), the activity of this enzyme decayed with a half-life of approximately 8 min. By use of rocket immunoelectrophoresis, indirect immunoprecipitation, and indirect immunoadsorption (coupled with pulse-chase experiments with 35S-labeled sulfate), the rapid initial loss in activity was shown to be due to enzyme inactivation rather than degradation of NADP-GDH antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained with anti-NADP-GDH immunoglobulin G showed that enzyme inactivation is accompanied by the conversion of enzyme subunits (Mr = 59,000) to a protein with a molecular weight of 118,000. Because this protein was stable during boiling and in the presence of sodium dodecyl sulfate and high concentrations of mercaptoethanol or dithiothreitol, it was tentatively assumed to be a covalently linked dimer of enzyme subunits. Pulse-chase experiments showed that total NADP-GDH antigen was subject to rapid degradation (t 1/2 = 88 min) in induced cells, and the same degradation rate was maintained after removal of ammonia from induced cells.

Chlorella↗

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

Ammonium Sulfate↗

Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells.

Ammonium Sulfate↗

Control of photorespiratory glycolate metabolism in an oxygen-resistant mutant of Chlorella sorokiniana.

Under a gas atmosphere of 99% O2/1% CO2, wild-type cells of Chlorella sorokiniana excreted 12% of their dry weight as glycolate during photolithotrophic growth, whereas mutant cells excreted glycolate at only 3% of the cellular dry weight. The observed difference in glycolate excretion by the two cell types appears to be due to a different capacity for the metabolism of glycolate, rather than to a different glycolate formation rate. This was concluded from experiments in which the metabolism of glycolate via the glycine-serine pathway was inhibited by the addition of isoniazid. Under such conditions, glycolate excretion rates for both cell types were identical. The mutant appeared to have significantly higher specific activities of glycine decarboxylase, serine hydroxymethyltransferase, serine-glyoxylate aminotransferase, glycerate kinase, and phosphoglycolate phosphatase than did the wild type. The specific activities of D-ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate dehydrogenase, glyoxylate-aminotransferase, and hydroxypyruvate reductase were the same for wild-type and mutant cells. The internal pool sizes of ammonia and amino acids increased in wild-type cells grown under high-oxygen concentrations but were hardly affected by high oxygen tensions in the mutant cells. Our results indicate that, under the growth conditions applied, the decarboxylation of glycine becomes the rate-limiting step of the glycine-serine pathway for the wild-type cells of C. sorokiniana.

Amino Acids↗

Novel salvage of queuine from queuosine and absence of queuine synthesis in Chlorella pyrenoidosa and Chlamydomonas reinhardtii.

Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7-deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.

Chlamydomonas↗

Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1.

We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.

Chlorella↗

Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1.

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.

Adenosine Monophosphate↗

Distinctive control of metabolic pathways by Chlorella autotrophica in semicontinuous culture.

The marine microalga Chlorella autotrophica was cultured semicontinuously under light-dark synchronizing conditions at two nutrient concentrations (2 and 4 mmol N.L-1) and five rates of daily renewal (from 10 to 50% of culture volume). Under such conditions, the biochemical composition of C. autotrophica was strongly influenced by the renewal rate, but unlike other marine microalgae, the nutrient concentration had no effect on the biochemical composition of the organic fraction of the microalga at a given growth rate. Results suggest that this species exerts a strong control over metabolic pathways, independent of the nutrient concentration in the medium.

Carbohydrates↗

Oral administration of hot water extracts of Chlorella vulgaris increases physical stamina in mice.

BACKGROUND/AIMS: A unicellular algae, Chlorella vulgaris, was used as a biological response modifier. Although hot water extracts of C. vulgaris (CVE) are thought to augment immune responses, the effect of CVE on fatigue and physical stamina has not been studied. METHODS: In the present study, we investigated the effect of CVE on forced swimming test and blood biochemical parameters related to fatigue, blood urea nitrogen (BUN), creatine kinase (CK), lactic dehydrogenase (LDH), glucose (Glc), and total protein (TP). CVE (0.05-0.15 g/kg/day) was orally administered to mice. RESULTS: After 7 days, the immobility time was decreased in the 0.1- and 0.15-g/kg CVE-treated groups (179 +/- 8.3 and 175 +/- 2.1 s) in comparison with the control group (223 +/- 5.4 s). In addition, the contents of BUN, CK, and LDH in the blood serum were decreased in the CVE-fed group. However, they had no effect on the elevation of Glc and TP level. CONCLUSIONS: The results predict a potential benefit of CVE for enhancing immune function and improving physical stamina.

Administration, Oral↗

Characterization of DNA polymerases in an uninfected and virus PBCV-1-infected green alga--Chlorella strain NC64A.

Chlorella NC64A cells infected with the large double-stranded DNA-containing virus PBCV-1 and uninfected cells were assayed for DNA polymerase activity. Both uninfected and infected cells contained three forms of DNA polymerase activity: (i) an exogenous DNA-dependent 10,000 g soluble fraction, (ii) an exogenous DNA-dependent 10,000 g particulate fraction, and (iii) a DNA-independent 10,000 g particulate fraction. The three DNA polymerase activities in the infected and uninfected cells were distinguished from one another by the conditions required for optimum activity and by their sensitivity to inhibitors.

Chlorella↗

Synthesis of the inner mitochondrial membrane and the intercalation of respiratory enzymes during the cell cycle of Chlorella.

The ratio of inner to outer mitochondrial membrane area remains close to 1-8 throughout the cell cycle in synchronized cells of Chlorella fusca var, vacuolata 211-8p. Using estimates of this ratio, together with our previous estimates of mitochondrial surface area, to calculate the absolute area of inner mitochondrial membrane, it is demonstrated that growth of the inner mitochondrial membrane during the cell cycle occupies an extended period and parallels the growth of the whole cell. In contrast, the synthesis of succinate dehydrogenase and cytochrome oxidase is restricted to the last third of the cell cycle. It is concluded that mitochondrial growth involves the intercalation of periodically synthesized respiratory enzymes into membranes made earlier in the cycle, with consequent 5-fold changes in the density of active enzyme molecules in the membrane. These observations are discussed in relation to the control of mitochondiral membrane synthesis, membrane assembly and respiration rate during the cell cycle.

Cell Division↗