Kinetics of β-N-methylamino-L-alanine (BMAA) and 2, 4-diaminobutyric acid (DAB) production by diatoms: the effect of nitrogen

The neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) are produced by cyanobacteria, diatoms and dinoflagellates and have been detected in seafood worldwide. Our present knowledge of their metabolism or biosynthesis is limited. In this study, the production of BMAA and DAB as a function of time was monitored in five strains representing four species of diatoms, i.e. Phaeodactylum tricornutum, Thalassiosira weissflogii, Thalassiosira pseudonana and Navicula pelliculosa, previously identified as BMAA and DAB producers. Subsequently, three strains were selected and exposed to three nitrogen treatments – starvation, control (the standard concentration in f/2 medium) and enrichment, because BMAA metabolism has been suggested to be closely associated with cellular nitrogen metabolism in both cyanobacteria and diatoms. Chlorophyll a and total protein concentrations were also determined. Our results indicate that BMAA and DAB production in diatoms is species- and strain-specific. However, production might also be affected by stress, particularly as related to nitrogen starvation and cell density. Furthermore, this study shows a significant correlation between the production of the two neurotoxins which might further suggest common steps in the metabolic pathways.     

UREASE GENE SEQUENCES FROM ALGAE AND HETEROTROPHIC BACTERIA IN AXENIC AND NONAXENIC PHYTOPLANKTON CULTURES1

While urea has long been recognized as an important form of nitrogen in planktonic ecosystems, very little is known about how many or which phytoplankton and bacteria can use urea as a nitrogen source. We developed a method, targeting the gene encoding urease, for the direct detection and identification of ureolytic organisms and tested it on seven axenic phytoplankton cultures (three diatoms, two prymnesiophytes, a eustigmatophyte, and a pelagophyte) and on three nonaxenic Aureococcus anophagefferens Hargraves et Sieburth cultures (CCMP1784 and two CCMP1708 cultures from different laboratories). The urease amplicon sequences from axenic phytoplankton cultures were consistent with genomic data in the three species for which both were available. Seven of 12 phytoplankton species have one or more introns in the amplified region of their urease gene(s). The 63 urease amplicons that were cloned and sequenced from nonaxenic A. anophagefferens cultures grouped into 17 distinct sequence types. Eleven types were related to α‐Proteobacteria, including three types likely belonging to the genus Roseovarius. Four types were related to γ‐Proteobacteria, including two likely belonging to the genus Marinobacter, and two types were related to β‐Proteobacteria. Terminal restriction fragment length polymorphism (TRFLP) analyses suggested that the sequenced amplicons represented approximately half of the diversity of bacterial urease genes present in the nonaxenic cultures. While many of the bacterial urease sequence types were apparently lab‐ or culture‐specific, others were found in all three nonaxenic cultures, suggesting the possibility of specific relationships between these bacteria and A. anophagefferens.     

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.     

CHRONIC EFFECTS OF ULTRAVIOLET-B RADIATION ON GROWTH AND CELL VOLUME OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cultures of the temperate estuarine diatom, Phaeodactylum tricornutum Bohlin (NEPCC Clone 31), were grown under ambient intensities of ultraviolet-A radiation (UVAR), photosynthetically active radiation (PAR), and various intensities of ultraviolet-B radiation (UVBR; 290–320 nm). Growth rates and cell volumes were monitored for 36 d. UVBR decreased growth rates and increased cell volumes. Sensitivity of growth to UVBR increased with time. Growth rates of P. tricornutum decreased with increasing ratios of UVBR:UVAR + PAR.     

Optimization of eicosapentaenoic acid production byPhaeodactylum tricornutumin the flat panel airlift (FPA) reactor

Biomass and eicosapentaenoic acid (EPA) productivities were investigated in a flat panel airlift loop reactor ideally mixed by static mixers. Growth with ammonium, urea and nitrate as nitrogen source were performed at different aeration rates. Cultures grew on ammonium but the decay of pH strongly inhibited biomass increase. On urea biomass productivity reached 2.35 g L^–1d^–1at an aeration rate of 0.66 vvm (24 h light per day, 1000 mol photon m^–2s^–1). Aeration rates between 0.33 vvm and 0.66 vvm and maximal productivities on urea were linearly dependent. Productivity on nitrate never exceeded 1.37 g L^–1d^–1. In the range of maximum productivity photosynthesis efficiency of 10.6% was reached at low irradiance (250 mol photon m^–2s^–1). Photosynthesis efficiency decreased to 4.8% at 1000 mol photon m^–2s^–1. At these high irradiances the flat panel airlift reactor showed a 35% higher volume productivity than the bubble column. At continuous culture conditions the influence of CO₂concentration in the supply air was tested. Highest productivities were reached at 1.25% (v/v) CO₂where the continuous culture yielded 1.04 g L^–1d^–1(16 h light per day, 1000 mol photon m^–2s^–1). The average EPA content amounted to 5.0% of cell dry weight, that resulted in EPA productivities of 52 mg L^–1d^–1(continuous culture, 16 h light per day) or 118 mg L^–1d^–1(batch culture, 24 h light per day).     

Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile

Mixotrophic growth of the eicosapentaenoic acid (EPA)producing diatom Phaeodactylum tricornutum UTEX640 was carried out in 1-L batch cultures under anexternal irradiance of 165 mol photons m^-2s^-1 by supplementing the inorganic culture mediumwith glycerol. The effect on the growth and the fattyacid profile was studied for different initialglycerol concentrations (0–0.1 M). The optimalglycerol concentration was 0.1 M.A lag phase was observed at high glycerolconcentrations. The present study also shows thatsuccessive additions of glycerol at 0.1M concentrationand using ammonium chloride as a nitrogen sourceremarkably increased the maximum biomass concentration(16.2 g L^-1) and maximum biomass productivity(61.5 mg L^-1 h^-1). These values wererespectively 9 and 8-fold higher than in thephotoautotrophically grown control. The level ofsaponifiable lipids in mixotrophically cultured cellswas significantly higher than in photoautotrophicallycultured cells and increased with the glycerolconcentration in the medium. The concentration ofstorage lipids, saturated and monounsaturated fattyacids, were enhanced but the EPA content did notchange significantly. The EPA content was around 2.2%of biomass dry weight. The maximum EPA yield was33.5 mg L^-1 d^-1 and was obtained in aculture containing 0.1 M glycerol, supplementedperiodically by ammonium chloride. This productivitywas 10-fold higher than the EPA productivity obtainedunder mixotrophic conditions.     

The secondary structure content of pigment-protein complexes from the thylakoids of two Chromophyte algae

The light-harvesting complex and Photosystem I have been isolated from thylakoids of two chromophyte algae by digitonin solubilisation and sucrose-gradient centrifugation. The -helic and β-structure content was determined by ultraviolet circular dichroism. The values obtained were approx. 40% -helix and approx. 14% β-structure for the light-harvesting complex of both Pavlova lutherii and Phaeodactylum tricornutum. For Photosystem I the values were approx. 55% -helix and 7% β-structure for both algae. It is concluded that for all photosynthetic antennae containing chlorophyll the dominant secondary structure is -helix.     

Effect of nitrate-nitrogen limitation on photosynthesis of the diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae)

Abstract Nitrate limited growth of the diatom Phaeodactylum tricornutum in chemostat cultures produced marked changes in biochemical composition and a six-fold reduction in the specific growth rate. This was associated with a reduction in the carbon and chlorophyll a specific light saturated rates, with little effect on light limited photosynthesis. Variations in specific growth rate were quantitatively related to carbon specific net photosynthesis and maximum chlorophyll a specific light saturated rates were positively correlated with cell nitrogen contents. The correlation between nitrogen content and photosynthesis for P. tricornutum and the differential effect of nitrogen supply on the light response curve of photosynthesis is qualitatively and quantitatively similar to published results for terrestrial vascular plants. There was little change in the photon (quantum) yield of photosynthesis which was not significantly different from 0.125mol O₂ mol photon^-1 the theoretical upper limit based on the Z scheme, even under severe nitrate deficiency. The capacity to maintain a high photon yield under nitrate limitation is discussed in relation to the nitrogen requirements of the stromal and membrane components of the photosynthetic apparatus.     

Phaeodactylum tricornutum: An established model species for diatom molecular research and an emerging chassis for algal synthetic biology

Diatoms are prominent and highly diverse microalgae in aquatic environments. Compared with other diatom species, Phaeodactylum tricornutum is an “atypical diatom” displaying three different morphotypes and lacking the usual silica shell. Despite being of limited ecological relevance, its ease of growth in the laboratory and well-known physiology, alongside the steady increase in genome-enabled information coupled with effective tools for manipulating gene expression, have meant it has gained increased recognition as a powerful experimental model for molecular research on diatoms. We here present a brief overview of how over the last 25 years P. tricornutum has contributed to the unveiling of fundamental aspects of diatom biology, while also emerging as a new tool for algal process engineering and synthetic biology.     

RESPONSE OF THE PHOTOSYNTHETIC APPARATUS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) TO NITRATE,PHOSPHATE, OR IRON STARVATION1

The effects of nitrate, phosphate, and iron starvation and resupply on photosynthetic pigments, selected photosynthetic proteins, and photosystem II (PSII) photochemistry were examined in the diatom Phaeodactylum tricornutum Bohlin (CCMP 1327). Although cell chlorophyll a (chl a) content decreased in nutrient-starved cells, the ratios of light-harvesting accessory pigments (chl c and fucoxanthin) to chl a were unaffected by nutrient starvation. The chl a-specific light absorpition coefficient (a*) and the functional absorption cross-section of PSII (σ) increased during nutrient starvation, consistent with reduction of intracellular self-shading (i.e. a reduction of the “package effect”) as cells became chlorotic. The light-harvesting complex proteins remained a constant proportion of total cell protein during nutrient starvation, indicating that chlorosis mirrored a general reduction in cell protein content. The ratio of the xanthophylls cycle pigments diatoxanthin and diadinoxanthin to chl a increased during nutrient starvation. These pigments are thought to play a photo-protective role by increasing dissipation of excitation energy in the pigment bed upstream from the reaction centers. Despite the increase in diatoxanthin and diadinoxanthin, the efficiency of PSII photochemistry, as measured by the ration of variable to maximum fluorescence (F_v/F_m) of dark-adapted cells, declined markedly under nitrate and iron starvation and moderately under phosphate starvation. Parallel to changes in F_v/F_m were decreases in abundance of the reaction center protein D1 consistent with damage of PSII reaction centers in nutrient-starved cells. The relative abundance of the carboxylating enzyme, ribulose bisphosphate carboxylase/oxygenase (RUBISCO), decreased in response to nitrate and iron starvation but not phosphate starvation. Most marked was the decline in the abundance of the small subunit of RUBISCO in nitrate-starved cells. The changes in pigment content and fluorescence characteristics were typically reversed within 24 h of resupply of the limiting nutrient.