Hiroya Araie

Selenium utilization strategy by microalgae.

The diversity of selenoproteins raises the question of why so many life forms require selenium. Selenoproteins are found in bacteria, archaea, and many eukaryotes. In photosynthetic microorganisms, the essential requirement for selenium has been reported in 33 species belonging to six phyla, although its biochemical significance is still unclear. According to genome databases, 20 species are defined as selenoprotein-producing organisms, including five photosynthetic organisms. In a marine coccolithophorid, Emiliania huxleyi (Haptophyta), we recently found unique characteristics of selenium utilization and novel selenoproteins using 75Se-tracer experiments. In E. huxleyi, selenite, not selenate, is the main substrate used and its uptake is driven by an ATP-dependent high-affinity, active transport system. Selenite is immediately metabolized to low-molecular mass compounds and partly converted to at least six selenoproteins, named EhSEP1–6. The most (EhSEP2) and second-most abundant selenoproteins (EhSEP1) are disulfide isomerase (PDI) homologous protein and thioredoxin reductase (TR) 1, respectively. Involvement of selenium in PDI is unique in this organism, while TR1 is also found in other organisms. In this review, we summarize physiological, biochemical, and molecular aspects of selenium utilization by microalgae and discuss their strategy of selenium utilization.

Identification and characterization of a selenoprotein, thioredoxin reductase, in a unicellular marine haptophyte alga, Emiliania huxleyi.

We found six selenoproteins (EhSEP1–6) in the coccolithophorid Emiliania huxleyi (Haptophyceae) using the 75Se radiotracer technique. Previously, the most abundant selenoprotein, EhSEP2, was identified as a novel selenoprotein, a protein disulfide isomerase-like protein (Obata, T., and Shiraiwa, Y. (2005) J. Biol. Chem. 280, 18462–18468). The present study focused on the second abundant selenoprotein, EhSEP1, in the same cells and analyzed its molecular properties and regulation of gene expression by selenium. The cDNA sequence of EhSEP1 consists of 1950 base pairs encoding a putative product of 495 amino acids with a calculated molecular mass of 52.2 kDa. The nucleotide and amino acid sequences of EhSEP1 showed strong similarities to those of the enzyme thioredoxin reductase (TR) 1 in the public databases. The EhSEP1 protein contains redox-active cysteine residues in the putative FAD binding domain of the pyridine nucleotide-disulfide oxidoreductase class-1 domain, a dimerization domain, and a C-terminal Gly-Cys-Sec (selenocysteine)-Gly sequence that is known to function as an additional redox center. In the 3′-untranslated region of EhSEP1 cDNA, we found a selenocysteine insertion sequence (SECIS) that is similar to the SECIS found previously in animals. The expression of EhSEP1 showed almost the same pattern under both selenium-sufficient and selenium-deficient conditions. Conversely, TR activity gradually increased 4-fold within ca. 70 h when cells were transferred to the medium containing 10 nm selenite. These data show that selenium is essential for the induction of TR activity at the translational level but not at the transcriptional level in this alga.

Proteomic analysis of lipid body from the alkenone‐producing marine haptophyte alga Tisochrysis lutea

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long‐chain (C37‐C40) ketones (alkenones) with 2–4 trans‐type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto‐fluorescence and no contamination of the most abundant cellular protein ribulose‐1,5‐bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC‐MS analysis, the LB can be more appropriately named as “alkenone body (AB).” Extracted AB‐containing proteins were analyzed by the combination of 1DE (SDS‐PAGE) and MS/MS for confident protein identification and annotated using BLAST tools at National Center for Biotechnology Information. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V‐ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone‐producing haptophytes, and a lipid raft‐associated SPFH domain‐containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER‐derived four layer‐envelopes chloroplast and function as the storage site of alkenones and alkenes.

Characterization of the Selenite Uptake Mechanism in the Coccolithophore Emiliania huxleyi (Haptophyta)

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ⁷⁵Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H⁺-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H⁺ concentration gradient may be a motive force for selenite transport. [⁷⁵Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO₃⁻, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H⁺-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.

Selenium in Algae

Selenium (Se) is an essential trace element for many organisms in order to be required for the synthesis of selenoproteins although it is very toxic at high concentration. More than 40 selenoprotein families have been identified in diverse organisms, including bacteria, archaea and eukaryotes. In photosynthetic microorganisms such as microalgae, many selenoproteins have also been identified by using bioinformatics approaches. Essentiality of Se requirement was experimentally proved in several algae, such as haptophyte algae, that possess selenoproteins. On the other hand, all of them that possess selenoproteins do not always show Se essential requirement in certain microalgae, such as a green algae. Se presents as four inorganic forms of selenide (−2), selenium (0), selenite (+2) and selenite (+4) and organic selenium-containing compounds. This Chapter focuses on the function of Se, membrane transport system, intracellular accumulation, metabolism to non-toxic organic compounds, and then the synthesis of selenoproteins involving a selenocysteine in which Se is replaced with sulfur. Especially, this Chapter introduces a unique property of E. huxleyi (coccolithophore, haptophyte) that possess two pathways for Se-compound production, namely both animal-like property to synthesize selenoproteins and land plant-like property to synthesize non-toxic organic compounds pathways. Algae can be considered to have evolved their properties for obtaining high viability by adjusting their Se-metabolism.

Characterization of a novel gene involved in cadmium accumulation screened from sponge-associated bacterial metagenome

Metagenome research has brought much attention for the identification of important and novel genes of industrial and pharmaceutical value. Here, using a metagenome library constructed from bacteria associated with the marine sponge, Styllisa massa, a high-throughput screening technique using radioisotope was implemented to screen for cadmium (Cd) binding or accumulation genes. From a total of 3301 randomly selected clones, a clone 247-11C was identified as harboring an open reading frame (ORF) showing Cd accumulation characteristics. The ORF, termed as ORF5, was further analyzed by protein functional studies to reveal the presence of a protein, Cdae-1. Cdae-1, composed of a signal peptide and domain harboring an E(G/A)KCG pentapeptide motif, enhanced Cd accumulation when expressed in Escherichia coli. Although showing no direct binding to Cd in vitro, the presence of important amino acid residues related to Cd detoxification suggests that Cdae-1 may possess a different mechanism from known Cd binding proteins such as metallothioneins (MTs) and phytochelatins (PCs). In summary, using the advantage of bacterial metagenomes, our findings in this work suggest the first report on the identification of a unique protein involved in Cd accumulation from bacteria associated with a marine sponge.

n-Nonacosadienes from the marine haptophytes Emiliania huxleyi and Gephyrocapsa oceanica

The hydrocarbons in cultures of marine haptophytes Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 were analyzed, and nonacosadienes and hentriacontadienes were detected as the major compounds in both strains. C29 and C31 monoenes and di-, tri- and tetra-unsaturated C33 alkenes were also detected as minor compounds but not C37 and C38 alkenes. The positions of the double bonds in the C29 and C31 alkenes were determined by mass spectrometry of their dimethyl disulfide (DMDS) adducts. Among the four C29 alkenes identified, the most abundant isomer was 2,20-nonacosadiene, and the other three compounds were 1,20-nonacosadiene, 3,20-nonacosadiene and 9-nonacosene, respectively. Hitherto, 2,20-nonacosadiene and 3,20-nonacosadiene were unknown to be natural products. The double bond at the n-9 (ω9) position in these C29 alkenes is hypothesized to be derived from precursors of unsaturated fatty acids possessing an n-9 double bond, such as (9Z)-9-octadecenoic acid. Nonacosadienes have the potential for being used as distinct haptophyte biomarkers.

Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme

Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry.

Changes in the accumulation of alkenones and lipids under nitrogen limitation and its relation to other energy storage metabolites in the haptophyte alga Emiliania huxleyi CCMP 2090

Alkenones are long-chain methyl/ethyl ketones (mainly in length of C37-C39) with two to four trans-unsaturated bonds produced by several kinds of marine haptophytes such as Emiliania huxleyi (coccolithophore). The physiological functions and metabolic profile of alkenones are not well known yet. In this study, we focused on elucidating how alkenones contribute to energy storage and cellular carbon partitioning in relation to other cellular components. For the purpose, we analyzed the changes in carbon allocation among various cell components like lipids, alkenones, proteins, and polysaccharides between cells exposed to N-sufficient (+N) and N-limited conditions (−N) in E. huxleyi CCMP 2090. Finally, the alkenones were found to function as main storage lipids and their accumulation was clearly increased by −N, whereas triacylglycerols (TAGs) were barely detected under any N conditions. The mobilization of carbons into alkenones was stimulated by −N from 15% under +N to 27% under −N. However, photosynthetic C allocation into other components was suppressed by −N, showing that percent C allocation into fatty acids, proteins, and polysaccharides was decreased from 9, 46, and 6.8% under +N to 7, 25, and 4.5% under −N, respectively. In addition, fatty acids such as 16:0, 18:0, 18:1, and 18:2 became dominant under −N while 18:5 became dominant under +N conditions, with no significant change in 22:6. This study revealed that alkenones function as primary carbon storage pools especially under −N condition in E. huxleyi CCMP 2090 and that N supply triggers a dynamic change in carbon metabolism by modifying membrane lipid composition and regulating carbon allocation preferences.