Diatom Phaeodactylum Tricornutum Bohlin Research Articles

SEQUENCE ANALYSIS AND TRANSCRIPTIONAL REGULATION OF IRON ACQUISITION GENES IN TWO MARINE DIATOMS1

The centric diatom Thalassiosira pseudonana Hasle et Heimdal and the pennate diatom Phaeodactylum tricornutum Bohlin possess genes with translated sequences homologous to high‐affinity ferric reductases present in model organisms. Thalassiosira pseudonana also possesses putative genes for membrane‐bound ferroxidase (TpFET3) and two highly similar iron (Fe) permeases (TpFTR1 and TpFTR2), as well as a divalent metal (M2+) transporter belonging to the NRAMP superfamily (TpNRAMP). In baker’s yeast, the ferroxidase–permease complex transports Fe(II) produced by reductases. We investigated transcript abundances of these genes as a function of Fe quota (QFe). Ferric reductase transcripts are abundant in both species (15%–60% of actin) under low QFe and are down‐regulated by 5‐ to 35‐fold at high QFe, suggesting Fe(III) reduction is a common, inducible strategy for Fe acquisition in marine diatoms. Permease transcript abundance was regulated by Fe status in T. pseudonana, but we did not detect significant differences in expression of the copper (Cu)‐containing ferroxidase. TpNRAMP showed the most dramatic regulation by QFe, suggesting a role in cellular Fe transport in either cell‐surface uptake or vacuolar mobilization. We could not identify ferroxidase or permease homologues in the P. tricornutum genome. The up‐regulation of genes in T. pseudonana that appear to be missing altogether from P. tricornutum as well as the finding that P. tricornutum seems to have an efficient system to acquire Fe′, suggest that diverse (and uncharacterized) Fe‐uptake systems may be at play within diatom assemblages. Different uptake systems among diatoms may provide a mechanistic basis for niche differentiation with respect to Fe availability in the ocean.

Regulation of the expression of intracellular beta-carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum.

Cells of the marine diatom Phaeodactylum tricornutum Bohlin (UTEX 642) grown in 5% CO(2) were transferred to air-level CO(2) in the light or dark and allowed to acclimate to air. No accumulation of the transcript of the P. tricornutum beta-carbonic anhydrase 1 (ptca1) was detected in 5% CO(2)-grown cells, but ptca1 mRNA accumulated and reached a peak after 6 h acclimation to air but decreased over the next 18 h. A similar accumulation time course was observed in cells air-acclimated in the dark, except that levels of mRNA were <50% those in the light. These results suggest that air-level [CO(2)] is required to trigger the transcription of ptca1 and that light affects the extent of acclimation. During acclimation to air for 120 h in the light, levels of ptca1 mRNA exhibited a periodic oscillation with a cycle of about 24 h, which, however, was not reflected in protein accumulation levels. A 5'-upstream region from the transcription-start site toward -1,292 bp of ptca1 was cloned by inverse polymerase chain reaction, and 5'-truncations were carried out on this fragment. The truncated promoter regions were fused with the beta-glucuronidase gene (uidA) and introduced into P. tricornutum. The promoter fragments, truncated at positions -1,292, -824, -484, -225, and -70 bp, conferred on transformants clear CO(2)-responsive beta-glucuronidase expressions. In contrast, the CO(2)-responsive regulation was severely impaired or completely abolished by truncations, respectively, at position -50 or -30 bp. These results indicate that critical cis-elements required for CO(2)-responsive transcription of ptca1 may be located between -70 and -30 bp relative to the transcription start site.

Identification and characterization of a new carbonic anhydrase in the marine diatom Phaeodactylum tricornutum

A cDNA encoding a new isoenzyme of β-type carbonic anhydrase (CA; EC 4.2.1.1) in the marine diatom Phaeodactylum tricornutum Bohlin has been cloned. The cDNA contained an open reading frame of 819 bp, which encodes a polypeptide of 273 amino acids. This gene, which is designated as ptca2, was found to be highly homologous (83% at the nucleotide level) to the previously isolated intracellular β-CA gene from Phaeodactylum tricornutum (ptca1). Comparison of the deduced amino acid sequence of ptca2 with β-CAs from other sources demonstrated that PtCA2 possesses the completely conserved zinc coordination residues of β-CA. The N-terminus 19 amino acid sequence of PtCA2 was predicted to be an endoplasmic reticulum-targeting signal, suggesting localization of the protein in an organelle or in the periplasmic space. Quantitative analysis of mRNA accumulation of ptca2 using real-time polymerase chain reaction revealed a significant level of mRNA accumulation even under 5% CO2 and a 3.5-fold increase in accumulation upon acclimation of the diatom to air. This indicates that ptca2 belongs to a constitutive class of enzyme that responds only weakly to the ambient CO2 concentration. The sequences of both ptca1 and ptca2 were shown to be grouped into a phylogeny that is composed of mixture of sequences from the eucarya and procarya domains, including sequences from the red alga Porphyridium purpureum, the green alga Coccomyxa, the red mold Neurospora crassa, and the yeast Saccharomyces cerevisiae.Key words: carbonic anhydrase, marine diatom, inorganic carbon concentrating mechanism (CCM), Phaeodactylum tricornutum.

Algal growth potential as an indicator of eutrophication degree in coastal areas under sewage disposal influence

Algal growth potential was used to quantify the degree of eutrophication of three coastal regions in São Paulo, Brazil which are subject to sewage disposal. Surface water was collected in Praia Grande, Santos, Guarujá and São Sebastião during two surveys (low tourist season—October 1997 and high tourist season—March 1998). Water was filtered and used in chlorophyll-a and nutrient analyses and in bioassays. Samples were inoculated with the diatom Phaeodactylum tricornutum Bohlin, and then subdivided into seven replicates. During a ten days experiment, in vivo fluorescence was performed and algal growth potential was calculated. Values were expressed as absolute in vivo fluorescence, as well as with a ratio of in vivo fluorescence of sample in relation to the fluorescence of control sea water for each treatment which defines the trophic status. The results of these bioassays showed a growing degree of eutrophication among regions: Praia Grande ∼ São Sebastião → Guarujá → Santos. It also identified sewage disposals and estuarine discharges as the main sources causing eutrophication. This bioassay technique is an important tool for environmental pre-monitoring studies, giving useful results in a short time.

ISOLATION OF ALGAL GENES BY FUNCTIONAL COMPLEMENTATION OF YEAST1

Algal cDNAs were isolated and characterized by functional complementation of yeast auxotrophs. Two cDNA libraries, one derived from the diatom Phaeodactylum tricornutum Bohlin and the other from the dinoflagellate Crypthecodinium cohnii Biecheler, were constructed using the Saccharomyces cerevisiae expression vector pFL-61. These libraries were used for functional complementation of auxotrophic markers in two yeast strains. Yeast tryptophan auxotrophs, complemented by the P. tricornutum library, contained a plasmid that encoded a two-domain protein associated with tryptophan synthesis, indole-3-glycerol phosphate synthase-N-(5′-phosphoribosyl) anthranilate isomerase. Another cDNA originating from the C. cohnii library rescued S. cervisiae from a defect in adenine biosynthesis. This cDNA encoded a fusion of phosphoribosylamidoimidazole-succinocarboxamide synthetase and phosphoribosylaminoimidazole carboxylase, which correspond to the yeast ade1 and ade2 genes, respectively. These results demonstrate that heterologous functional complementation can be used to identify algal genes and may provide advantages over other gene discovery methods.

EFFECT OF OLIGOMYCIN ON DARK RESPIRATION IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE): IMPLICATIONS FOR DETERMINATION OF MAINTENANCE RESPIRATION

Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O2 consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed.

CHARACTERIZATION OF A GENE ENCODING THE LIGHT-HARVESTING VIOLAXANTHIN-CHLOROPHYLL PROTEIN OF NANNOCHLOROPSIS SP. (EUSTIGMATOPHYCEAE).

In contrast to vascular plants, green algae, and diatoms, the major light-harvesting complex of the marine eustigmatophyte genus Nannochloropsis is a violaxanthin-chlorophyll a protein complex that lacks chlorophylls b and c. The isolation of a single polypeptide from the light-harvesting complex of Nannochloropsis sp. (IOLR strain) was previously reported (Sukenik et al. 1992). The NH2 -terminal amino acid sequence of this polypeptide was significantly similar to NH2 -terminal sequences of the light-harvesting fucoxanthin, chlorophyll a/c polypeptides from the diatom Phaeodactylum tricornutum Bohlin. Using polyclonal antibodies raised to the Nannochloropsis light-harvesting polypeptide, a gene encoding this polypeptide was isolated from a cDNA expression library. The deduced amino acid sequence of the Nannochloropsis violaxanthin-chlorophyll a polypeptide reveals a 36 amino acid presequence followed by 173 amino acids that constitute the mature polypeptide. The mature polypeptide has 30%-40% sequence identity to the diatom fucoxanthin-chlorophyll a/c polypeptides and less then 27% identity to the green algal and vascular plant light-harvesting chlorophyll polypeptides that bind both chlorophylls a and b. Its molecular mass, as deduced from the gene sequence, is 18.4 kDa with three putative transmembrane helices and several residues that may be involved in chlorophyll binding. The cDNA encoding the violaxanthin-chlorophyll a polypeptide was used to isolate and characterize a 10 kb genomic fragment containing the entire gene. The open reading frame was interrupted by five introns ranging in size from 123 to 449 bp. The intron borders have typical eukaryotic GT … AG sequences.

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha‐T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcpA promoter. pPha‐T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat‐1 genes confer resistance to the antibiotic nourseothricin, and nptII confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uidA and gfp were also introduced into P. tricornutum using pPha‐T1. Gus expression in some transformants reached 15 μg·μg−1 of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum, enhancing the potential of this organism for exploring basic biological questions and industrial applications.

Immunolocalization and distribution of form ii rubisco in the pyrenoid and chloroplast stroma of amphidinium carterae and form i rubisco in the symbiont‐derived plastids of peridinium foliaceum (dinophyceae)

Chloroplasts of peridinin‐containing dinoflagellates have recently been shown to contain Form II Rubisco, which consists of large subunits only and is coded by nuclear genes. We have used immunoelectron microscopy to determine the distribution of Form II and Form I Rubisco in dinoflagellates. In sections of Amphidinium carterae Hulburt, the pyrenoid was intensely labeled and the rest of the chloroplast moderately labeled by antisera to Form II Rubisco from the purple non‐sulfur bacterium Rhodospirillum rubrum and the symbiotic dinoflagellate Symbiodinium sp. No labeling was observed when sections were exposed to antiserum against Form I Rubisco of the haptophyte alga Isochrysis galbana. In contrast, cell sections of the dinoflagellate Peridinium foliaceum (Stein) Biecheler, whose chloroplasts belong to a diatom endosymbiont, showed no labeling with the two antisera against Form II Rubisco, but heavy pyrenoid labeling was present after treatment with antiserum against Form I Rubisco of I. galbana. The same immunolabeling results were obtained with the free‐living diatom Phaeodactylum tricornutum Bohlin. Volumetric analysis of the distribution of Form II Rubisco in the chloroplast of A. carterae showed that, in cells grown under moderate photon irradiance, 72.9% of the plastid's Rubisco was localized in the pyrenoid, whereas in cells grown under low irradiance only 37.0% of the Rubisco was found in the pyrenoid. This light‐induced concentration of Rubisco in the pyrenoid suggests that a CO2–concentrating mechanism may elevate CO2 within the pyrenoid, favoring the efficient fixation of CO2 by pyrenoid Rubisco.

Effect of Cadmium on Growth, ATP Content, Carbon Fixation and Ultrastructure in the Marine Diatom Phaeodactylum tricornutum Bohlin

Cadmium toxicity to the diatom Phaeodactylum tricornutum Bohlin has been studied on the basis of the effect of this metal on growth, ATP content, 14C uptake and cellular ultrastructure. The microalga was exposed to 1, 5, 10, 25, 50, 75 and 100 mg l-1 of cadmium and showed a concentration-dependent inhibition in those physiological parameters. At cadmium concentrations of 5 mg l-1 or higher, a significant effect on growth of P. tricornutum was observed. After 8 hours of exposure to the different cadmium concentrations, the decrease in the ATP content was significant only at cadmium concentrations of 25 mg l-1 or higher. 14C uptake was more susceptible to cadmium than the decrease in the ATP content, as 5 mg l-1 of cadmium caused a severe decrease in this parameter. Cadmium also caused ultrastructural changes in P. tricornutum cells: deposition of cadmium on the surface of cell, increase in the chloroplast size, appearance of electrodense granulations, and reduction in lipid inclusions.

ACCUMULATION OF FERREDOXIN AND FLAVODOXIN IN A MARINE DIATOM IN RESPONSE TO FE

Despite recognition that Fe availability is significant in regulating oceanic production in some regions, the biogeochemistry of this trace element is poorly understood. To complement contemporary methods of analytical chemistry, we have used an immunological approach to monitor the Fe nutrition of marine phytoplankton. In prokaryotes and numerous microalgae, the redox catalyst ferredoxin is functionally replaced by flavodoxin during periods of Fe deficiency. In this study, antibodies were raised against ferredoxin purified from a marine diatom, and their utility as a diagnostic indicator was assessed. A species survey demonstrated broad reactivity with both pennate and centric diatoms and additionally with several nondiatom taxa. In batch cultures of the diatom Phaeodactylum tricornutum Bohlin, in which Fe levels were varied, accumulation of ferredoxin varied with the physiological state of the culture; in unimpaired cells (Fv/Fm≥ 0.65), ferredoxin levels were high, whereas levels dropped markedly in cells experiencing even slight photochemical impairment. Accumulation of flavodoxin varied inversely with that of ferredoxin. An experiment was performed to demonstrate the temporal pattern of accumulation of ferredoxin upon recovery from Fe limitation. Prior to Fe amendment, cells were physiologically impaired (chlorotic, Fv/Fm &lt; 0.3) and contained flavodoxin but no detectable ferredoxin. Following addition of Fe, constraints on photochemistry were relaxed within hours. Coinciding with this, levels of flavodoxin declined, whereas ferredoxin was accumulated to high levels within 8 h.

Steady-states of semicontinuous cultures of a marine diatom: Effect of saturating nutrient concentrations

The evolution of nitrogen incorporation, the C–N ratio and chlorophyll content were studied in semicontinuous, light–dark synchronised cultures of the marine diatom Phaeodactylum tricornutum Bohlin, maintained under conditions of limiting to highly saturating nutrient concentrations and at different dilutions. The behaviour of cultures diverged from the expected since a decrease in the nitrogen cellular quota and a stable chlorophyll a–C ratio was recorded under nutrient-sufficient conditions, indicating that current theories on continuous cultures cannot be applied directly to semicontinuous cultures when maintained under highly nutrient-saturated conditions.

MEASURING RATES OF AMMONIUM ASSIMILATION IN MARINE ALGAE: USE OF THE PROTONOPHORE CARBONYL CYANIDE m‐CHLOROPHENYLHYDRAZONE TO DISTINGUISH BETWEEN UPTAKE AND ASSIMILATION

A method for determining rates of ammonium assimilation in marine algae is described. Ammonium assimilation is defined as the decrease in total (medium + cellular) ammonium. The protonophore carbonyl cyanide m‐chlorophenylhydrazone (CCCP) was used to distinguish between uptake and assimilation of ammonium. Ammonium uptake by nitrogen‐replete and nitrogen‐starved cells of the diatom Phaeodactylum tricornutum Bohlin and the green macroalga Enteromorpha sp. was completely (98%–99%) inhibited in the presence of 100 μM CCCP. In addition to inhibiting further uptake of ammonium, CCCP promoted the release of unassimilated ammonium by nitrogen‐replete and nitrogen‐starved P. tricornutum and Enteromorpha that had been allowed to take up ammonium for a period. Most (97.5%) of preaccumulated 14C‐methylammonium was released by nitrogen‐starved P. tricornutum in the presence of CCCP. Specific rates of ammonium assimilation in nitrogen‐replete cultures of P. tricornutum were identical to the maximum growth rate, but specific rates in nitrogen‐starved cultures were fourfold greater. Rates of ammonium assimilation in Enteromorpha during both the surge and the internally controlled uptake phases were the same as the internally controlled rate of uptake, suggesting that the latter is a reliable measure of the maximum rate of assimilation.

Removal of cadmium ions by the marine diatom Phaeodactylum tricornutum Bohlin accumulation and long-term kinetics of uptake

The marine diatom Phaeodactylum tricornutum Bohlin was exposed to different cadmium concentrations (1–100 mg l −1) for 4 days. The amount of cadmium removed was recorded, with particular attention paid to long-term uptake kinetics, and to the cellular location of cadmium. Cadmium accumulation occurred at all concentrations assayed. The EC 50 of cadmium to P. tricornutum was 22·39 mg l −1 after 4 days of exposure. Cadmium uptake followed a saturation kinetic at cadmium concentrations ≥25 mg l −1. However, at lower cadmium concentrations, the uptake of this metal followed a linear trend for all days of culture. At cadmium concentrations in the medium lower than 25 mg l −1, P. tricornutum removed cadmium mainly within the cell. At higher cadmium concentrations, the amount of cadmium removed by adsorption to the cell surface was higher than intracellular cadmium, because of the toxic effects of cadmium on P. tricornutum cells. This toxicity reduced the cadmium accumulation within the cells.

Flow cytometric analysis of nocodazole-induced cell-cycle arrest in the pennate diatom Phaeodactylum tricornutum Bohlin

The microtubule inhibitor, nocodazole (2.5 mg L-1), can arrest the cell cycle of the pennate diatom Phaeodactylum tricornutum Bohlin at G2 + M phase. Flow cytometric analysis of cells treated with nocodazole suggest that the proportion of cells at G2 + M phase can accumulate to over 95%. Even after a 48-h treatment with nocodazole (2.5 mg L-1), the cells can still exit mitosis, suggesting that the cell-cycle arrest is reversible.

Long-chain class III metallothioneins as a mechanism of cadmium tolerance in the marine diatom Phaeodactylum tricornutum Bohlin

Cadmium tolerance of the marine diatom Phaeodactylum tricornutum Bohlin was studied. P. tricornutum growth was significantly reduced by cadmium concentrations of 5 mg l −1 or higher. The EC 50 value was 22.39 mg l −1 of cadmium after four days of exposure. Therefore, P. tricornutum is more tolerant to cadmium than other microalgal cells. In response to exposure to cadmium, P. tricornutum synthesize class III metallothioneins which sequester the metal in a harmless form. However, cadmium tolerance of P. tricornutum cells is not only due to the ability of this microalga to synthesize class III metallothioneins (γ-Glu-Cys) n -Gly, but also because these metallothioneins are composed of long-chain polypeptides. The cadmium-metallothionein complexes of P. tricornutum cells contain predominantly polypeptides of n = 4−9. Long-chain metallothioneins are more efficient in binding heavy metals. Capillary electrophoresis was used to separate class III metallothioneins. This is a recent technique used to separate many molecules. With this technique, the length of the separated polypeptides can also be estimated.

Dissolved inorganic carbon utilization and the development of extracellular carbonic anhydrase by the marine diatom Phaeodactylum tricornutum.

The presence of extracellular carbonic anhydrase (CA) in relation to medium composition was investigated using cultures of the marine diatom Phaeodactylum tricornutum Bohlin. Large-volume cultures, with low initial cell inocula were grown on ASP-2 (no dissolved inorganic carbon (DIC), 550 μM NOa - ), f/2 (2-0 mM DIC, 880μM NO3 - ) and modified f/2 (2.0 mM DIC, 20 μM NO3 - media. Cells growing on ASP-2 showed extracellular CA in the early stages of growth, whereas extracellular CA was not detected until partial depletion of total DIC in the stationary phase for cultures on f/2 or modified f/2. Both HCO3 - and CO2 were important in carbon limitation, extracellular CA being present when the free-CO2 concentration fell below 5 μM, but the HCO3 - concentration needed to be below 1 mM. When carbon-replete cells were transferred to carbon-limited conditions, extracellular CA was recorded within minutes, the process being light-dependent and completely inhibited by 3,3,4-dichlorophenyl-l, 1-dimethylurea (DCMU). The addition of DIC to carbon-limited cells resulted in a rapid decrease in extracellular CA activity. The membrane-impermeable inhibitor of carbonic anhydrase, dextran-bound sulphonamide (DBS) was used to inhibit extracellular CA activity in relation to photosynthetic rate in carbon-replete and carbon-limited cells. At the lowest DIC concentration (O'lOniM), for cells with maximum external CA activity, DBS gave over 80% inhibition of the photosynthetic rate, demonstrating the key role of external CA in maintaining high photosynthetic rate under conditions of carbon limitation. It is proposed that the key factor in the regulation of extracellular CA activity is the total Hux of inorganic carbon (C.) into the cell. This determines the Ci , flux into the chloroplast and when this is inadequate to support the photosynthetic rate attained by a carbon-replete chloroplast at optimum photon flux density, extracellular CA is activated. This mechanism would explain the observed interaction of CO2 and HCO3 - in the regulation of extracellular CA activity.

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

ABSTRACTThe effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen‐starved in the presence of sodium. The carbon‐to‐nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen‐ and sodium‐deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.

Eicosapentaenoic acid-rich biomass production by the microalga Phaeodactylum tricornutum in a continuous-flow reactor

The marine diatom Phaeodactylum tricornutum Bohlin is a potential source of the pharmaceutically valuable ω3 polyunsaturated fatty acid eicosapentaenoic acid (EPA). The results of indoor continuous growth of Phaeodactylum tricornutum Bohlin are reported. The relationships between dilution rate ( D), nitrate concentration and chemical composition were studied. Higher biomass and lipid productivities were obtained at low D values. EPA was found to be an intermediate metabolite and the best productivity (6 mg l −1 day −1 was achieved for D values ranging from 0·32 to 0·50 day −1. Under optimum conditions, 84 and 11%, respectively, of total recovered EPA were present in monogalactosyldiacylglycerol (MGDG) and in triacylglycerol (TG) moieties, respectively. Recorded EPA/AA and EPA/20·4 ω3 ratios for all tested dilution rates were among the highest values ever reported, showing EPA purification to be easier to perform from this starting material than from many others commonly in use.

In Situ Glutamine Synthetase Activity in a Marine Unicellular Alga (Development of a Sensitive Colorimetric Assay and the Effects of Nitrogen Status on Enzyme Activity).

A malachite green colorimetric assay for glutamine synthetase is described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodactylum tricornutum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-N[prime]-2-ethanesulfonic acid compared with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Activities were higher in cells permeabilized in the presence of reductant, particularly dithiothreitol. Glutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. In the presence of saturating concentrations of glutamate and ATP, the apparent Km for ammonia was 320 [mu]M, but this value decreased to 110 [mu]M with subsaturating concentrations of glutamate and ATP. The apparent Km values for glutamate and ATP, in the presence of saturating concentrations of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cells had lower glutamine synthetase activities than did nitrate-grown cells. During nitrogen starvation of both ammonia- and nitrate-grown cells, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time course for the increases in glutamine synthetase activities and rate of methylamine uptake following the transfer of nitrate-grown cells to nitrogen-deficient medium were very similar. In nitrate-grown cells and cells deprived of combined nitrogen, glutamine synthetase activities and maximum rates of ammonia uptake gave comparable values when measured at the same temperature (20[deg]C).