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PubMed1987

Characterization of Iron Uptake from Ferrioxamine B by Chlorella vulgaris.

Iron uptake from two Fe(3+)-hydroxamate siderophores, ferrioxamine B and Fe(3+)-rhodotorulate, by iron-stressed Chlorella vulgaris (ATCC strain 11468) was evaluated with some comparison to iron uptake from synthetic and organic acid ferric chelates. Iron-stress induced iron uptake from ferrioxamine B. Dissipation of the electrochemical gradient, via uncouplers, inhibited iron uptake. Respiratory inhibitors gave variable results, an indication that a direct link to respiration was not apparent. Vanadate inhibition of iron uptake indicated that an ATPase or phosphate intermediate could be involved in the uptake mechanism. Divalent cations manifested variable effects dependent on the cation and chelator used. These data confirm that C. vulgaris has an inducible iron-uptake system for Fe(3+)-hydroxamic acid siderophores which may involve a different mechanism than that observed for other chelates.

Journal Article↗
PubMed1987

Evaluation of Reductive Release as a Mechanism for Iron Uptake from Ferrioxamine B by Chlorella vulgaris.

The involvement of ferric reduction in the iron uptake mechanism of iron-stressed Chlorella vulgaris from ferrioxamine B was investigated. Some comparative data for ferric-citrate was also obtained. EPR and a spectrophotometric assay were used to measure Fe(3+) reduction. These two methods differed in the absolute quantity but not in effectors of ferric reduction. The mechanism governing ferric reduction was investigated by use of respiratory inhibitors, uncouplers, alternative electron acceptors, and ATPase inhibitors. Reduction appears to play a role in iron uptake from both Fe(3+)-deferrioxamine B and Fe(3+)-citrate; however, the involvement of photoreduction in Fe(3+)-citrate uptake implies multiple reductive mechanisms could be involved.

Journal Article↗
PubMed1988

Biosynthesis of Tetrapyrrole Pigment Precursors : Formation and Utilization of Glutamyl-tRNA for delta-Aminolevulinic Acid Synthesis by Isolated Enzyme Fractions from Chlorella Vulgaris.

The universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) is formed from glutamate (Glu) in algae and higher plants. In the postulated reaction sequence, Glu-tRNA is produced by a Glu-tRNA synthetase, and the product serves as a substrate for a reduction step catalyzed by a pyridine nucleotide-requiring Glu-tRNA dehydrogenase. The reduced intermediate is then converted into ALA by a transaminase. An RNA and three enzyme fractions required for ALA formation from Glu have been isolated from soluble Chlorella extracts. The recombined fractions catalyzed ALA production from Glu or Glu-tRNA. The fraction containing the synthetase produced Glu-tRNA from Glu and tRNA in the presence of ATP and Mg(2+). The isolated product of this reaction served as substrate for ALA production by the partially reconstituted enzyme system lacking the synthetase fraction and incapable of producing ALA from Glu. The production of ALA from Glu-tRNA by this partially reconstituted system did not require free Glu or ATP, and was not affected by added ATP. These results show that (a) free Glu-tRNA is an intermediate in the formation of ALA from Glu, (b) ATP is required only in the first step of the reaction sequence, and NADPH only in a later step, (c) Glu-tRNA production is the essential reaction catalyzed by one of the enzyme fractions, (d) this enzyme fraction is active in the absence of the other enzymes and is not required for activity of the others. The specific Glu-tRNA synthetase required for ALA formation has an approximate molecular weight of 73,000 +/- 5,000 as determined by Sephadex G-100 gel filtration and native polyacrylamide gel electrophoresis. Other Glu-tRNA synthetases were present in the cell extracts but were ineffective in the the ALA-forming process.

Journal Article↗
PubMed1989

Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock.

Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24 degrees C to 4 degrees C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24 degrees C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before.

Journal Article↗
PubMed1989

Biosynthesis of Tetrapyrrole Pigment Precursors : Pyridoxal Requirement of the Aminotransferase Step in the Formation of delta-Aminolevulinate from Glutamate in Extracts of Chlorella vulgaris.

The aminotransferase that catalyzes the formation of delta-aminolevulinic acid from glutamate-1-semialdehyde or from glutamate in a reconstituted enzyme system was isolated and partially purified from Chlorella vulgaris. The apparent molecular weight of the aminotransferase was determined by Sephadex G-100 and Ultrogel AcA 54 gel filtration to be 60,000 +/- 5,000. Catalytic activity of the aminotransferase required pyrixodal phosphate (PALP). The cofactor could not be removed by gel filtration after exposure of the enzyme to PALP. Aminotransferase was inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The concentration of gabaculine required for half maximal inhibition was about 0.05 micromolar. Aminotransferase activity could be regained upon the removal of gabaculine by gel filtration and supplementing the assay medium with PALP. Neither the inhibitory action of gabaculine nor its reversibility was affected by preincubation of the enzyme with the keto acids levulinate and delta-aminolevulinic acid.

Journal Article↗
PubMed1990

Uptake of Inorganic Carbon by Isolated Chloroplasts of the Unicellular Green Alga Chlorella ellipsoidea.

Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO(2) and PGA-dependent rates of O(2) evolution of 64.5 to 150 micromoles per milligram of chlorophyll per hour, that is 30 to 70% of the photosynthetic activity of the parent cells. Intact chloroplasts showed no carbonic anhydrase activity, but it was detected in preparations of ruptured organelles. Rates of photosynthesis, measured in a closed system at pH 7.5, were twice the calculated rate of CO(2) supply from the uncatalyzed dehydration of HCO(3) (-) indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO(2) depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO(2) transport across the chloroplast envelope.

Journal Article↗
PubMed1990

Acetohydroxy Acid Synthase Activity in Chlorella emersonii under Auto- and Heterotrophic Growth Conditions.

Acetohydroxyacid synthase (AHAS) activity was studied in the green unicellular alga Chlorella emersonii. This activity and its regulation was compared in the algae grown autotrophically and heterotrophically on glucose in the dark. No evidence for the existence of more than one enzyme was found. The activity in crude extracts from either heterotrophically or autotrophically grown cells showed a K(m) for pyruvate of 9 millimolar, a 22-fold preference for 2-ketobutyrate over pyruvate as the second substrate, 50% inhibition by 0.5 millimolar valine, and 50% inhibition by 0.3 micromolar sulfometuron methyl (SMM). Spontaneous mutants of the alga resistant to SMM were isolated, which appeared to be single gene mutants containing SMM-resistant AHAS activity. Hence, AHAS appears to be the sole direct target site of SMM in C. emersonii. The fact that the mutants had equivalent SMM resistance under auto- and heterotrophic conditions further supports the conclusion that the same enzyme functions under both physiological regimes. The addition of valine and isoleucine leads to partial relief of SMM inhibition of biomass increase, but not of SMM inhibition of cell division.

Journal Article↗
PubMed1990

Regulation of Chloroplast Development by Nitrogen Source and Growth Conditions in a Chlorella protothecoides Strain.

Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6-bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6-phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media.

Journal Article↗
PubMed1997

Crystallization of the RNA guanylyltransferase of Chlorella virus PBCV-1.

RNA guanylyltransferase, or capping enzyme (E.C. 2.7.7.50) catalyzes the transfer of GMP from GTP to diphosphate-terminated RNA to form the cap structure GpppN. Chlorella virus capping enzyme expressed in E. coli has been purified, treated with GTP and crystallized. X-ray diffraction data have been collected from these crystals as well as for a mercury derivative obtained by soaking the crystals in thimerosal. Selenomethionine RNA guanylyltransferase was purified and crystallized in a similar fashion. The space group is C2221 and the cell parameters are a = 93.3, b = 214.9, c = 105.8 A. Two Hg atoms and two subsets of Se atoms have been localized using difference Patterson and Fourier methods, suggesting that there are two molecules per asymmetric unit.

Journal Article↗
PubMed1968

Respiratory inhibition in Chlorella produced by "purified" polyethylene glycol 1540.

Polyethylene glycol 1540, added to culture solution in amounts sufficient to reduce the water potential to -10 atmospheres, inhibited respiration in Chlorella much more than mannitol solutions at -10 atmospheres. This occurred despite the purification of the polyethylene glycol by passage through exchange columns. The toxic properties, which developed some time after purification, increased with time of storage of solutions of polyethylene glycol 1540 at room temperature.

Depression, Chemical↗
PubMed1966

Gas exchange of algae. II. Effects of oxygen, helium, and argon on the photosynthesis of Chlorella pyrenoidosa.

Changes in the oxygen partial pressure of air over the range of 8 to 258 mm of Hg did not adversely affect the photosynthetic capacity of Chlorella pyrenoidosa. Gas exchange and growth measurements remained constant for 3-week periods and were similar to air controls (oxygen pressure of 160 mm of Hg). Oxygen partial pressures of 532 and 745 mm of Hg had an adverse effect on algal metabolism. Carbon dioxide consumption was 24% lower in the gas mixture containing oxygen at a pressure 532 mm of Hg than in the air control, and the growth rate was slightly reduced. Oxygen at a partial pressure of 745 mm of Hg decreased the photosynthetic rate 39% and the growth rate 37% over the corresponding rates in air. The lowered metabolic rates remained constant during 14 days of measurements, and the effect was reversible after this time. Substitution of helium or argon for the nitrogen in air had no effect on oxygen production, carbon dioxide consumption, or growth rate for 3-week periods. All measurements were made at a total pressure of 760 mm of Hg, and all gas mixtures were enriched with 2% carbon dioxide. Thus, the physiological functioning and reliability of a photosynthetic gas exchanger should not be adversely affected by: (i) oxygen partial pressures ranging from 8 to 258 mm of Hg; (ii) the use of pure oxygen at reduced total pressure (155 to 258 mm of Hg) unless pressure per se affects photosynthesis, or (iii) the inclusion of helium or argon in the gas environment (up to a partial pressure of 595 mm of Hg).

Argon↗
PubMed1967

Gas exchange of algae. 3. Relation between the concentration of carbon dioxide in the nutrient medium and the oxygen production of Chlorella pyrenoidosa.

The oxygen production of a photosynthetic gas exchanger containing Chlorella pyrenoidosa (1% packed cell volume) was measured when various concentrations of carbon dioxide were present within the culture unit. The internal carbon dioxide concentrations were obtained by manipulating the entrance gas concentration and the flow rate. Carbon dioxide percentages were monitored by means of electrodes placed directly in the nutrient medium. The concentration of carbon dioxide in the nutrient medium which produced maximal photosynthesis was in the range of 1.5 to 2.5% by volume. Results were unaffected by either the level of carbon dioxide in the entrance gas or the rate of gas flow. Entrance gases containing 2% carbon dioxide flowing at 320 ml/min, 3% carbon dioxide at 135 ml/min, and 4% carbon dioxide at 55 ml/min yielded optimal carbon dioxide concentrations in the particular unit studied. By using carbon dioxide electrodes implanted directly in the gas exchanger to optimize the carbon dioxide concentration throughout the culture medium, it should be possible to design more efficient large-scale units.

Carbon Dioxide↗
PubMed1968

Gas exchange of algae. IV. Reliability of Chlorella pyrenoidosa.

A single culture of Chlorella pyrenoidosa (700 ml) was grown continuously under uniform environmental conditions for a period of 11 months. During this time, the culture remained uncontaminated and its oxygen production, carbon dioxide consumption, and photosynthetic quotient (PQ = CO(2)/O(2)) were monitored on a 24-hr basis. The gas exchange characteristics of the alga were found to be extremely reliable; the average oxygen production was 1.21 +/- 0.03 ml per min, the carbon dioxide consumption was 1.09 +/- 0.03 ml per min, and the PQ was 0.90 +/- 0.01 when changes in both lamp intensity and instrument accuracy were taken into consideration. Such long-term dependability in the production of oxygen, consumption of carbon dioxide, and maintenance of a uniform PQ warrants the use of C. pyrenoidosa in a regenerative life support system for space travel.

Automation↗
PubMed1969

Oxygen-tolerant strain of chlorella sorokiniana.

An oxygen-tolerant strain (OTS) of Chlorella sorokiniana was isolated by growing and continuously subculturing the oxygen-sensitive strain (OSS) in growth medium bubbled continuously with 95% O(2)-5% CO(2). Under these conditions, 6 to 8 hr were required before the OSS began to grow. The growth rates of the OTS and the OSS were the same when grown in an atmosphere of air-5% CO(2), and the growth rate of the OTS was the same when aerated with 95% O(2)-5% CO(2) and air-5% CO(2). The adaptive process was irreversible since serial transfers of the OTS grown with air-5% CO(2) did not alter the ability of the OTS to grow with 95% O(2)-5% CO(2). Inasmuch as photosynthesis in the OTS and the OSS was inhibited to a similar extent by oxygen and the adaptive process occurred heterotrophically in the absence of photosynthesis, it was concluded that the adaptive process was independent of photosynthesis. Morphological differences between the OTS and the OSS are presented and discussed with respect to the adaptive process.

Adaptation, Biological↗
PubMed1969

Numerical Taxonomy of Heterotrophic Bacteria Growing in Association with Continuous-Culture Chlorella sorokiniana.

Twenty-nine cultures isolated from nonaxenic growths of Chlorella sorokiniana (Shihira and Krauss) and eight reference cultures were tested for 150 morphological and biochemical characteristics. The taxonomic data were subjected to computer analysis from which five major clusters were identified. The bacterial isolates have been placed in the genera: Pseudomonas, Acinetobacter, Flavobacterium, and Bacillus. Amino acid requirements of four strains from the test set were determined.

Journal Article↗
PubMed1997

Interactive effects of UV-B and Cu on photosynthesis, uptake and metabolism of nutrients in a green alga Chlorella vulgaris under simulated ozone column.

This study demonstrated a general reduction in photosynthesis (carbon fixation, O(2)-evolution and photochemical electron transport chain), the uptake of NH(4)(+), NO(3)(-), urea and PO(4)(3+), and activities of nitrate reductase, urease, acid phosphatase and ATPase following UV-B and copper exposure of Chlorella vulgaris in the absence or presence of 1 and 2 ppm concentrations of a 4-inch-thick ozone layer. Though the effect of stressors used in combination was very detrimental to the above processes, selected concentrations of ozone not only counteracted the UV-B-induced inhibition of the above processes, but also stimulated O(2)-evolution and the photochemical electron transport chain. Kinetics of nutrient uptake and enzyme activities demonstrated that UV-B causes structural change(s) in the enzymes/carriers responsible for the uptake of NH(4)(+), NO(3)(-), urea and PO(4)(3+) as well as their assimilatory enzymes. Except for nitrate reductase, copper was found to compete for the binding sites of all the above enzymes. Synergistic inhibition of photosynthetic activity, nutrient (except NH(4)(+)) uptake, and enzyme activities by UV-B+Cu seems to be due to increased Cu uptake as a consequence of altered membrane permeability brought about by the peroxidation of membrane lipids in UV-B-exposed cells.

Journal Article↗