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PubMed1992

Cloning, sequencing, overproduction, and purification of M. CviBI (GANTC) methyltransferase from Chlorella virus NC-1A [corrected].

We have cloned and sequenced the cvibIM gene from Chlorella virus NC-1A by selecting for the modification phenotype. The modification gene was cloned on a 7-kb BamHI fragment inserted into the BamHI site of the pUC13 plasmid. The cvibIM gene was localized at the 3' end of this fragment. Sequencing of this region revealed a large open reading frame that codes for methyltransferase (MTase; symbol M.) (predicting 260 amino acids). M.CviBI (GANTC) aa sequence is homologous to M.Dam(GATC), M.DpnII(GATC), and M.T4 (GATC), and not so to M.HinfI(GANTC), M.HhaII (GANTC), and M.DpnA(GATC). We also describe the use of the polymerase chain reaction technique to alter transcriptional and translational signals surrounding this gene so as to achieve overexpression in Escherichia coli. This construct yields M.CviBI at 2-3% of the total cellular protein. The MTase was purified by phosphocellulose, DEAE, and gel filtration chromatography. Its size by SDS-PAGE is approx. 28 kDa, in good agreement with that predicted from the nucleotide sequence.

Amino Acid Sequence↗
PubMed1992

Characterization of the gene encoding the most abundant in vitro translation product from virus-infected Chlorella-like algae.

The gene (33kDa) encoding a 33-kDa peptide from Chlorella virus, PBCV-1, was cloned and sequenced. This gene encodes the most abundant in vitro translation product synthesized from viral mRNAs isolated beginning at 20 min post-infection. The message persisted throughout the remainder of the viral life cycle. An open reading frame (ORF) of 717 bp, which encodes a polypeptide of 238 amino acids with a predicted M(r) or 26,613, was found on a 2752-bp cloned fragment from PBCV-1 HindIII restriction fragment 9. Transcriptional analysis of this ORF indicated that it was expressed both early and late, and as the viral life cycle progressed, the mRNA increased in size and abundance. Three other ORFs were also found; the largest of which (741 bp) hybridized to a low-abundance transcript which would encode a polypeptide with a predicted M(r) of 27,854.

Amino Acid Sequence↗
PubMed1995

Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella.

The gene (cviJIR) encoding the two/three-base R.CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R.CviJI required overexpression of the gene encoding the M.CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R.CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced 'star' activity (R.CviJI cleaves at RGCY, while R.CviJI* also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R.CviJI. The very high frequency of DNA cleavage by R.CviJI* was exploited in the development of a quasi-random shotgun library method. R.CviJI*-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.

Amino Acid Sequence↗
PubMed1994

Characterization of the nuclear large-subunit rRNA-encoding gene and the group-I self-splicing intron from Chlorella ellipsoidea C-87.

We found two group-I self-splicing introns in both the large subunit (LSU) and small subunit (SSU) of the nuclear rRNA-encoding genes (rDNA) of the unicellular green alga, Chlorella ellipsoidea C-87 (Ce). The primary and secondary structures of the LSU rRNA (3350 nt) and its intron (445 nt) were characterized. The intron was inserted in the conserved stem 32 of the LSU rRNA and contained all P1-P10 motifs of the group-IB intron. In vitro transcripts of the LSU rDNA containing the intron sequence displayed a strong self-splicing activity at high salt concentrations. The overall structure and splicing conditions of the LSU rRNA intron were, however, considerably different from those of the SSU rRNA intron of the same organism. These results suggest different origins and/or different evolutionary courses of these Ce introns.

Base Sequence↗
PubMed1996

Cloning and characterization of the nitrate reductase-encoding gene from Chlorella vulgaris: structure and identification of transcription start points and initiator sequences.

The reduction of nitrate to nitrite catalyzed by nitrate reductase (NR) is considered to be the rate-limiting and regulated step of nitrate assimilation, a major metabolic pathway occurring in a wide range of organisms which in turn supply the nutritional nitrogen requirements for other forms of life. Chlorella vulgaris NR mRNA levels are very responsive to changes in nitrogen source. In the presence of ammonia as the sole nitrogen source, under repressed conditions, NR mRNA is undetectable. Under inducing conditions, the removal of ammonia and addition of nitrate, rapid NR mRNA synthesis occurs. We are studying the elements involved in regulating the expression of this important gene. Two overlapping genomic clones (NRS1 and NR5') were isolated from a cosmid library. The two clones were sequenced and their sequences were aligned with that of a full-length NR cDNA. The gene is approximately 8 kb long and consists of 19 exons and 18 introns. Unlike NR isolated from other species, the exons which code for the functional domains of C. vulgaris are separated by introns. Two transcription start points (tsp) were identified and each is surrounded by potential initiator sequences. No TATA, CAAT or GC-rich promoter elements were located. A time course of NR induction revealed that while transcription initiation from one tsp remains at a constant level from the point of induction through steady state, the level of initiation from another tsp is high upon induction, but decreases as steady state is attained.

Amino Acid Sequence↗
PubMed1989

A mimic of superoxide dismutase activity protects Chlorella sorokiniana against the toxicity of sulfite.

The spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been used to demonstrate the light-dependent production of O2- by Chlorella sorokiniana. In the presence of SO3= a light-dependent production of the sulfur trioxy anion radical (SO3-.) could also be seen. A complex prepared by reacting desferrioxamine with MnO2, which catalyzes the dismutation of O2-, protected the alga against the toxicity of sulfite. The data suggest that SO2 toxicity is at least partially due to the effects of sulfoxy-free radicals generated by the oxidation of SO3= by O2-.

Chlorella↗
PubMed2005

Impact of zinc acclimation on bioaccumulation and homeostasis in Chlorella kesslerii.

Growth curves, cellular Zn contents and cellular protein expression were examined for the green alga, Chlorella kesslerii, as a function of different Zn growth regimes (growth in 16 pM, 1.7 nM or 1.6 microM calculated Zn2+). Zn homeostasis was responsible for observed differences in the capacity of the organism to accumulate Zn. The rapid acclimation that occurred in response to a Zn deficiency was likely due to the production of Zn transport sites. No differences were observed among cellular phytochelatin contents or efflux rate constants, although efflux did play an important role in regulating Zn cellular content. A long-term adaptation to Zn was not thought to occur since bioaccumulation and biological responses were similar for four successive cultures (30-40 days, 16-19 cell cycles) at different [Zn2+]. Among proteins that were influenced by the Zn growth regime, the Rubisco and histone H3 proteins were identified as being induced in the presence of 1.6 microM Zn2+ as compared with 1.7 nM Zn2+. The impact of the Zn preconditioning demonstrated that the concentrations of essential metals in the algal growth media would have an important, if not predominant effect on toxicity or bioaccumulation assessments. Furthermore, the high regulation of Zn transport and intracellular events by the microorganisms will likely preclude the use of simple metal uptake models including the free ion activity model and the biotic ligand model to predict either bioaccumulation or toxicological effects of Zn and perhaps other essential metals.

Blotting, Western↗
PubMed2006

Bioaccumulation of cadmium in an experimental aquatic food chain involving phytoplankton (Chlorella vulgaris), zooplankton (Moina macrocopa), and the predatory catfish Clarias macrocephalus x C. gariepinus.

The accumulation of cadmium (Cd) was studied in an experimental aquatic food chain involving the phytoplankton Chlorella vulgaris as the primary producer, the zooplankton Moina macrocopa as the primary consumer, and the catfish Clarias macrocephalus x Clarias gariepinus as the secondary consumer. C. vulgaris was first exposed to Cd solutions at 0.00, 0.35, and 3.50 mg l(-1), referred to as control group and experimental groups 1 and 2, respectively. Subsequently, each group was fed to three corresponding groups of M. macrocopa. Finally, three groups of catfish were fed these corresponding groups of M. macrocopa. After C. vulgaris was exposed to 3.50 mg l(-1) Cd (experimental group 2), the residual Cd in solution was only 4.01 microg l(-1), lower than the maximum allowable limit of Cd in natural water sources (5 microg l(-1)). Cd concentrations in C. vulgaris were 0.01+/-0.00 microg g(-1) (dry wt) in the control group, 194+/-1.80 microg g(-1) (dry wt) in experimental group 1, and 1140+/-20.06 microg g(-1) (dry wt) in experimental group 2. The Cd concentrations in M. macrocopa were 0.01+/-0.00 microg g(-1) (dry wt) in the control group, 16.48+/-2.23 microg g(-1) (dry wt) in experimental group 1, and 56.6+/-3.23 microg g(-1) (dry wt) in experimental group 2. The Cd concentrations in catfish muscle increased with increasing Cd concentrations in the food. After 60 days of fish culture, the mean concentrations of Cd in fish muscle were 0.01+/-0.00 microg g(-1) (dry wt) in the control group, 0.61+/-0.02 microg g(-1) (dry wt) in experimental group 1 and 1.04+/-0.06 microg g(-1) (dry wt) in experimental group 2. Cd concentration in fish muscle of experimental group 2 was equal to the permissible limit. Cd accumulation affected fish growth: at the end of the study, the mean fresh weight (12.81 g) of catfish in the control group, was significantly higher than those experimental group 1 (10.43 g) and experimental group 2 (10.00 g). The results showed that the measurement of Cd concentration in water does not necessarily give a measure of the safety of aquatic organisms as human food. Hence, heavy metal contamination is a matter for concern when organisms are harvested, for fish and human consumption, from natural water sources.

Animals↗
PubMed2004

Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae.

A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.

Alkaline Phosphatase↗
PubMed2005

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa.

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.

Animals↗
PubMed2005

Effect of Chlorella pyrenoidosa on fecal excretion and liver accumulation of polychlorinated dibenzo-p-dioxin in mice.

The effect of Chlorella pyrenoidosa on fecal excretion and liver accumulation of polychlorinated dibenzo-p-dioxin in C57BL/6N mice administered dioxin was examined. Mice were administered 2.2 microg of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (H6CDD) dissolved in corn oil once after a period of acclimatization, after which they were fed either a basal diet, a 10% C. pyrenoidosa diet, or a 10% Spinach diet, for five weeks. Among mice fed the 10% C. pyrenoidosa diet, cumulative fecal excretion of H6CDD over the first week following administration was significantly greater (9.2-fold) than that observed among mice fed the basal diet. Moreover, excretion during the fifth week following administration of H6CDD was still significantly greater (3.1-fold) among mice fed the 10% C. pyrenoidosa diet than among mice fed the basal diet. Five weeks after administration of H6CDD, liver accumulation of H6CDD in mice fed the 10% C. pyrenoidosa diet was significantly less than that observed among mice fed either the basal diet and the Spinach diet (by 27.9% and 34.8%, respectively). These findings suggest that C. pyrenoidosa may be useful in inhibiting the absorption of dioxins via food and the reabsorption of dioxins stored already in the body in the intestinal tract, thus preventing accumulation of dioxins within the body.

Animals↗
PubMed2005

The effect of solvents on metal ion adsorption by the alga Chlorella vulgaris.

The dead dried alga, Chlorella vulgaris, was used for metal ion sequestering. The uptake of each of Cu(II), Cd(II), Fe(III), and Sn(IV) from their aqueous solutions decreased upon reuse of the biomass. Introducing mixed ethanol/water (50% v/v) metal ion solutions in batch systems enhanced the metal uptake of the exhausted biomass by 90% for iron, 40% for tin and only 14% for cadmium. In the column system, 20% v/v ethanol/water proved to be superior to acetone/water and isopropanol/water metal solutions in enhancing metal uptake. This increase in uptake offers a new method for regeneration of the algal capacity to remove metal ions from their solutions. Mossbauer results of iron and tin loaded algae from aqueous and mixed ethanol water solutions exhibited the characteristic doublet for (FeCl4-) at 0.350-0.565 mms(-1) respectively and the major doublet for inorganic Sn(IV) moieties at -0.217 to -0.365 mms(-1) respectively, excluding the possibility of microprecipitation of iron and tin, which is expected at such high concentrations of metals in the alga.

Adsorption↗
PubMed2005

Improved high performance liquid chromatographic method for determination of carotenoids in the microalga Chlorella pyrenoidosa.

Microalgae have become an important commercial source of carotenoids and microalgae-derived functional foods are consumed by people worldwide. Therefore, an HPLC method was developed to discern the variety and content of carotenoids in the microalga Chlorella pyrenoidosa. The microalga sample was powdered, extracted, saponified and subjected to HPLC analysis. A mobile phase of methanol-acetonitrile-water (84:14:2, v/v/v) (A) and methylene chloride (100%) (B) with the following gradient elution was developed: 100% A and 0% B in the beginning, maintained for 14 min, decreased to 95% A in 25 min, 75% A in 30 min, 74% A in 35 min, 45% A in 50 min and returned to 100% A in 55 min. A total of 32 carotenoids were resolved within 49 min by using a C30 column with flow rate at 1 mL/min and detection at 450 nm. An internal standard beta-apo-8'-carotenal was used to quantify all the carotenoids. All-trans-lutein was present in exceptionally large amount (125034.4 microg/g), followed by cis isomers of lutein (27975.3 microg/g), all-trans-alpha-carotene (2465.8 microg/g), zeaxanthin (2170.3 microg/g), cis isomers of beta-carotene (2159.3 microg/g), all-trans-beta-carotene (2155.0 microg/g), cis isomers of alpha-carotene (1766.7 microg/g), beta-cryptoxanthin (334.9 microg/g), neoxanthin and its cis isomers (199.7 microg/g), neochrome (65.2 microg/g), auroxanthin (38.5 microg/g) and violaxanthin and its cis isomers (38.1 microg/g).

Carotenoids↗
PubMed2004

Differential sensitivity of two green algae, Scenedesmus quadricauda and Chlorella vulgaris, to 14 pesticide adjuvants.

Growth-inhibition tests for 14 pesticide adjuvants which are widely used to manufacture various pesticidal formulations in China, were performed on the green algae Scenedesmus quadricauda, and Chlorella vulgaris to compare differential sensitivity among populations of these algae to the adjuvants. The results showed that the acute toxicities of 700#, 1601#, By-140, and SOPA to S. quadricauda and C. vulgaris were the lowest among all the tested adjuvants. The acute toxicities of Tween 80, O-25, and AEO-13 phosphate to the selected two green algae were intermediate among the tested adjuvants. The acute toxicities of 602#, 500#, OT, NP-10, OP-10, and JFC were the highest. Meanwhile, the algal species vary widely in their response to those adjuvants. The results showed that there was a differential response to various adjuvants among the selected algal species and that the sensitivities of the various algal species exposed to 1601# and OT varied by nearly 1 order and to 700#, and By-140 varied by over 1 order of magnitude. In addition, the NOEC and LOEC values of the selected 14 adjuvants to S. quadricauda and C. vulgaris were tested; the result showed that NOEC < or = EC10<EC20 for 27 of the 28 data sets (96%) and EC10 < or = LOEC < or = EC20 for 20 of the 28 data sets (78%).

Chlorella↗
PubMed2004

Impact of nitrophenols on the photosynthetic electron transport chain and ATP content in Nostoc muscorum and Chlorella vulgaris.

Concentration-dependent inhibition of the photosynthetic electron transport chain (photosystem I (PS I), photosystem II (PS II) and whole chain reaction) and ATP content was observed in Nostoc muscorum and Chlorella vulgaris grown with o-nitrophenol, m-nitrophenol, or 2,4-dinitrophenol. Although the extents of inhibition of the photosynthetic electron transport chain in both organisms were similar, PS II was more sensitive than PS I and whole chain reaction to the nitrophenols. Depletion of the ATP pool was noted in nitrophenol-grown cultures, probably as a consequence of nearly complete inhibition of the photosynthetic electron transport chain.

2,4-Dinitrophenol↗
PubMed2004

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.

Antimycin A↗
PubMed2005

Cultivation factors and population size control the uptake of nitrogen by the microalgae Chlorella vulgaris when interacting with the microalgae growth-promoting bacterium Azospirillum brasilense.

Growth of and the capacity to take up nitrogen in the freshwater microalgae Chlorella vulgaris were studied while varying the concentrations of ammonium and nitrate, the pH and the source of carbon in a synthetic wastewater growth medium when co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense. Analyses of 29 independent experiments showed that co-immobilization of the microalgae with A. brasilense could result in two independent phenomena directly affected by cultivation factors, such as nitrogen species, pH and presence of a carbon source. First, growth of the microalgal population increased without an increase in the capacity of the single cells to take up nitrogen, or second, the capacity of cells to take up nitrogen increased without an increase of the total microalgal population. These phenomena were dependent on the population density of the microalgae, which was in turn affected by cultivation factors. This supports the conclusion that the size of the microalgal population controls the uptake of nitrogen in C. vulgaris cells - the higher the population (regardless the experimental parameters), the less nitrogen each cell takes up.

Azospirillum brasilense↗
PubMed2004

High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides.

The use of renewable energy sources is becoming increasingly necessary to mitigate global warming. Recently much research has been focused on identifying suitable biomass species, which can provide high-energy outputs, to replace conventional fossil fuels. This paper reports an approach for increasing the yield of bio-oil production from fast pyrolysis after manipulating the metabolic pathway in microalgae through heterotrophic growth. The yield of bio-oil (57.9%) produced from heterotrophic Chlorella protothecoides cells was 3.4 times higher than from autotrophic cells by fast pyrolysis. The bio-oil was characterized by a much lower oxygen content, with a higher heating value (41 MJ kg(-1)), a lower density (0.92 kg l(-1)), and lower viscosity (0.02 Pas) compared to those of bio-oil from autotrophic cells and wood. These properties are comparable to fossil oil. The research could contribute to the creation of a system to produce energy from microalgae, and also could have great commercial potential for liquid fuel production.

Chlorella↗