Enzymatic conversion of glutamate to delta-aminolevulinate in soluble extracts of the unicellular green alga, Chlorella vulgaris

Cell-free preparations from the unicellular green alga, Chlorella vulgaris, catalyze the conversion of glutamate to delta-aminolevulinate, which is the first committed step in heme and chlorophyll biosynthesis. Most activity remains in the supernatant fraction after centrifugation at 264,000g. Additional activity can be solubilized from the high-speed pellet by treatment with 0.5 M NaCl. After gel filtration through Sephadex G-25, the reaction catalyzed by the high-speed supernatant requires glutamate, ATP, Mg2+, and NADPH. Boiled extract is inactive. The pH optimum is between 7.8 and 7.9 and the temperature optimum is 30 degrees C. Concentrations required for half-maximal activity are 0.05 mM glutamate, 0.4 mM ATP, 6 mM MgCl2, and 0.4 mM NADPH or 0.7 mM NADH. The reaction requires no additional amino donor. Involvement of pyridoxal phosphate in the catalytic mechanism is suggested by sensitivity to pyridoxal antagonists; 50% inhibition is achieved with 5 microM gabaculine or 0.4 mM aminooxyacetate. Involvement of two or more enzymes is suggested by the nonlinear reaction rate dependence on protein concentration. Evidence for the involvement of an activated glutamate intermediate was obtained by product formation after sequential addition and removal of substrates, and by inhibition (80%) with 1 mM hydroxylamine. Protoheme inhibits the activity by 50% at 1.2 microM. Preincubation of the extract with ATP causes stimulation and/or stabilization of the activity compared to preincubation without ATP or no preincubation. In preparations obtained from C. vulgaris strain C-10, which requires light for greening, dark-grown cells yield one-third as much activity as 4-h-greened cells.     

Copper tolerance in the green alga, Chlorella vulgaris

Abstract. The effect of sub-lethal concentrations of copper upon tolerant and non-tolerant strains of Chlorella vulgaris was investigated. Copper concentrations of 0.2 and 0.4 mg dm⁻³ increased the lag phase of both strains, the effect being greater in the non-tolerant strain. No difference was observed in the toxicity of copper to the photosynthetic rates of the isolated chloroplasts of either strain. However, significant differences were shown at the whole cell level.
Lower copper uptake was shown by the tolerant cells. In both strains initial uptake of copper was followed by a phase of desorption before cell division occurred. In cultures of both strains the concentration of ionic copper was decreased by complexation with extracellular organic material. Over a 14 day growth period more organic material was produced by the tolerant cells. The organic material produced by the tolerant cell formed organo-copper complexes which had a higher conditional stability constant.
It is proposed that the cell wall acts as a barrier to copper in the tolerant cells and prevents copper from affecting cell metabolism. Organo-copper complexation occurs at this barrier and this complex is then released into the medium.     

A new inducible expression system in a transformed green alga, Chlorella vulgaris

Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.     

Augmentation of antitumor resistance by a strain of unicellular green algae,Chlorella vulgaris

Summary Growth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different.     

Basis of the trade-off between parasitoid resistance and larval competitive ability in Drosophila melanogaster

Drosophila melanogaster can be artificially selected for increased resistance against parasitoid wasps that attack the larvae. Lines selected for greater resistance are poorer larval competitors under conditions of resource scarcity. Here we investigated the mechanistic basis of this apparent trade-off. We found that resistant lines have approximately twice the density of haemocytes (blood cells) than that of controls. Haemocytes are involved in encapsulation, the chief cellular immune defence against parasitoids. We have previously shown that resistant lines feed more slowly than controls and hypothesize that limiting resources are being switched from trophic to defensive functions.     

Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 degrees C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 degrees C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 degrees C, respectively, with only 33 and 23% of maximal activities being retained at 10 degrees C. In presence of 2 microM flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 degrees C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 degrees C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 degrees C (Q10) and apparent activation energy (E(a)) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 degrees C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Lack of trade-off between direct and indirect defence against grape powdery mildew in riverbank grape

Abstract.  1. The hypothesis that plants that invest more in an indirect defence will invest less in direct mechanisms was tested using genotypes of the riverbank grape Vitis riparia that varied in expression of acarodomatia, tufts of non-glandular trichomes on underside of leaves that are frequently occupied by mycophagous mites. Mycophagous mites in this system have been shown to protect vines from grape powdery mildew caused by the fungus Uncinula necator .
2. Multiple cane cuttings from 24 genotypes of V. riparia , a species native to North America, were obtained from the USDA grape genetics repository and planted out in a common garden.
3. Genotypes varied in size of acarodomatia by threefold and varied in density of mycophagous mites by over 10-fold. Genotypes originating from the western part of the distribution had smaller acarodomatia and fewer mycophagous mites than genotypes from the eastern part of the distribution. There was a positive genetic correlation between acarodomatia size and mite density.
4. In a separate experiment, where vines were kept free of mycophagous mites, genotypes showed 10-fold variation in percentage leaf surface colonised by mildew. There was, however, no relationship between mildew on leaves and size of acarodomatia. Mildew did not vary depending on origin of genotype, although female vines tended to have greater levels than males.
5. No evidence was found for a trade-off between investment in an indirect defence mediated through acarodomatia and investment in direct defence against grape powdery mildew. Mite/plant defence mutualisms are contrasted with ant/plant defence mutualisms where there is better evidence for trade-offs under field conditions.     

Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana

Wild type (WT), and nitrate reductase (NR)- and nitrite-reductase (NiR)-deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate-grown WT cells was very low in air, increased slightly after addition of nitrite (200 microM), but strongly under anoxia. Importantly, even completely NR-free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR-free cells. In contrast, NiR-deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.     

Potential trade-off between vocal ornamentation and spatial ability in a songbird

Bird song is hypothesized to be a reliable indicator of cognition because it depends on brain structure and function. Song features have been found to correlate positively with measures of cognition, but the relationship between song and cognition is complicated because not all cognitive abilities are themselves positively correlated. If cognition is not a unitary trait, developmental constraints on brain growth could generate trade-offs between some aspects of cognition and song. To further clarify the relationship between song and cognition in song sparrows (Melospiza melodia), we examined repertoire size and performance on a spatial task. We found an inverse relationship between repertoire size and speed of spatial learning and suggest that a developmental trade-off between the hippocampus and song control nuclei could be responsible for this relationship. By attending to male song, females may learn about a suite of cognitive abilities; this study suggests that females may glean information about a male''s cognitive weaknesses as well as his strengths.     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

Competition and succession between the oily alga Botryococcus braunii and two green algae Chlorella vulgaris and Chlamydomonas reinhardtii

Contamination by other algal species is a major problem in the culture of the oil-producing green alga Botryococcus braunii (Bb). In this study, the population interactions between Bb and two small green algae, Chlorella vulgaris (Cv) and Chlamydomonas reinhardtii (Cr), were separately investigated in two controlled experiments. Filtrates of Cv or Cr significantly stimulated the growth of Bb in the first 9–11 days when the filtrates were derived from a low- or medium-density algal culture. However, filtrates of Cv and Cr from the high-cell-density cultures significantly inhibited Bb growth from day 11 and 7, respectively. In Bb–Cv competition, the growth of Bb was significantly inhibited by Cv at the biomass ratio of Bb/Cv?=?1:1. However, when the Cv component reduced to 0.5 (Bb/Cv?=?1:0.5) or 0.25 (Bb/Cv?=?1:0.25), the growth of Bb was faster than that in the control in the first 13 days. Similarly, in Bb–Cr competition, the biomass ratios of 1:0.5 and 1:0.25 (Bb/Cr) favoured the growth of B. braunii in the first 15 days, but the growth of Bb decreased in the Bb/Cr ratio of 1:1 from day 15. By contrast, the growth of Cv and Cr were not affected by the presence of Bb in all biomass ratios. These results suggest that Cv and Cr could impact the growth of Bb by metabolites in the culture medium, and when the biomass of Bb is two to four times that of other algae, this oily alga becomes competitive over other algal species. This study reveals the mechanism of population dynamics of this oily alga competing with small and fast-growing algae. The results are applicable in reducing algal competition and regulating species succession between slow and fast-growing algae species.     

Jasmonic acid affects changes in the growth and some components content in alga Chlorella vulgaris

The present study was undertaken to test the influence of exogenous applied jasmonic acid upon the growth and changes in some metabolites levels in the cells of green alga Chlorella vulgaris Beijerinck (Chlorophyceae). It was found, that JA in algal cells acted in a concentration-dependent manner. Treatment with JA at high concentrations range of 10⁻⁵–10⁻⁴ M, resulted in the decrease in cell number and reduction of major photosynthetic pigments, monosaccharides, soluble cellular and extracellular proteins levels as well as decrease in pH of the medium. In contrast to 10⁻⁵ and 10⁻⁴ M JA, this phytohormone applied at 10⁻⁸–10⁻⁶ M induced the increase in cell number, photosynthetic pigments and monosaccharides contents, significant accumulation and extracellular secretion of soluble proteins over control and neutralization of the medium. Quantitative changes in polypeptide pattern of total cellular proteins after treatment with the optimal concentration of 10⁻⁷ M JA on the 7^th day of cultivation as analyzed by SDS-PAGE, was also observed. JA induced synthesis de novo of 15 specific polypeptides with molecular weight 334-16 kDa which were’t detected in the control. The data suggest that JA plays a important role in algal growth and development.     

Nitrogen,phosphorus and high CO2 modulate photosynthesis,biomass and lipid production in the green alga Chlorella vulgaris

Photosynthesis Research - Climate change could impact nutrient bioavailability in aquatic environment. To understand the interaction of nutrient bioavailability and elevated CO2, Chlorella vulgaris...     

No trade-off between learning ability and parasitoid resistance in Drosophila melanogaster

Learning ability and immunity to parasites are linked at the physiological level in several insect species. The aim of this work was to investigate the relationship between learning and immunity at an evolutionary level. We tested whether selection for improved learning ability in Drosophila melanogaster led to changes in parasitoid resistance as a correlated response. Similarly, we assayed whether selection for better parasitoid resistance led to a change in learning ability. There was no significant difference between selected and control lines in either case; the estimated confidence intervals for the differences indicate that a trade-off relationship is unlikely.     

Effect of brassinosteroids on growth and proton extrusion in the alga Chlorella vulgaris Beijerinck (Chlorophyceae)

Brassinolide, as a plant hormone, promotes growth of a number of plant species. Similar effects are induced by its epimer 24-epibrassinolide. In this paper we discuss the effects of brassinosteroids on the growth and proton extrusion in the green alga Chlorella vulgaris (Chlorophyceae). At concentrations between 10^–15 and 10^–8 m, brassinolide and 24-epibrassinolide induce a significant stimulation of growth and H⁺ extrusion. The growth was associated with an increase in the capability of algal cells to acidify the medium, where brassinolide is biologically more active than 24-epibrassinolide.Abbreviations BL brassinolide - BR(s) brassinosteroid(s) - epiBL 24-epibrassinolide - DW dry weight - IAA indole-3-acetic acid