Kimberlee Thamatrakoln

Silicon Uptake in Diatoms Revisited: A Model for Saturable and Nonsaturable Uptake Kinetics and the Role of Silicon Transporters

The silicic acid uptake kinetics of diatoms were studied to provide a mechanistic explanation for previous work demonstrating both nonsaturable and Michaelis-Menten-type saturable uptake. Using 68Ge(OH)4 as a radiotracer for Si(OH)4, we showed a time-dependent transition from nonsaturable to saturable uptake kinetics in multiple diatom species. In cells grown under silicon (Si)-replete conditions, Si(OH)4 uptake was initially nonsaturable but became saturable over time. Cells prestarved for Si for 24 h exhibited immediate saturable kinetics. Data suggest nonsaturability was due to surge uptake when intracellular Si pool capacity was high, and saturability occurred when equilibrium was achieved between pool capacity and cell wall silica incorporation. In Thalassiosira pseudonana at low Si(OH)4 concentrations, uptake followed sigmoidal kinetics, indicating regulation by an allosteric mechanism. Competition of Si(OH)4 uptake with Ge(OH)4 suggested uptake at low Si(OH)4 concentrations was mediated by Si transporters. At high Si(OH)4, competition experiments and nonsaturability indicated uptake was not carrier mediated and occurred by diffusion. Zinc did not appear to be directly involved in Si(OH)4 uptake, in contrast to a previous suggestion. A model for Si(OH)4 uptake in diatoms is presented that proposes two control mechanisms: active transport by Si transporters at low Si(OH)4 and diffusional transport controlled by the capacity of intracellular pools in relation to cell wall silica incorporation at high Si(OH)4. The model integrates kinetic and equilibrium components of diatom Si(OH)4 uptake and consistently explains results in this and previous investigations.

COMPARATIVE SEQUENCE ANALYSIS OF DIATOM SILICON TRANSPORTERS: TOWARD A MECHANISTIC MODEL OF SILICON TRANSPORT†

Silicon is an important element in biology, for organisms ranging from unicellular algae to humans. It acts as a structural material for both plants and animals, but can also function as a metabolite or regulator of gene expression, affecting a wide range of cellular processes. Molecular details of biological interaction with silicon are poorly understood. Diatoms, the largest group of silicifying organisms, are a good model system for studying this interaction. The first proteins shown to directly interact with silicon were diatom silicon transporters (SITs). Because the basis for substrate recognition lies within the primary sequence of a protein, identification of conserved amino acid residues would provide insight into the mechanism of SIT function. Lack of SIT sequences from a diversity of diatoms and high sequence conservation in known SITs has precluded identification of such residues. In this study, PCR was used to amplify partial SIT sequences from eight diverse diatom species. Multiple gene copies were prevalent in each species, and phylogenetic analysis showed that SITs generally group according to species. In addition to partial SIT sequences, full‐length SIT genes were identified from the pennate diatom, Nitzschia alba (Lewin and Lewin), and the centric diatom Skeletonema costatum (Greville) Cleve. Comparing these SITs with previously identified SITs showed structural differences between SITs of centrics and pennates, suggesting differences in transport mechanism or regulation. Comparative amino acid analysis identified conserved regions that may be important for silicon transport, including repeats of the motif GXQ. A model for silicon uptake and efflux is presented that is consistent with known aspects of transport.

Analysis of Thalassiosira pseudonana Silicon Transporters Indicates Distinct Regulatory Levels and Transport Activity through the Cell Cycle

ABSTRACT An analysis of the expression and activity of silicon transporters (SITs) was done on synchronously growing cultures of the diatom Thalassiosira pseudonana to provide insight into the role these proteins play in cellular silicon metabolism during the cell cycle. The first SIT-specific polyclonal peptide antibody was generated and used in the immunoblot analysis of whole-cell protein lysates to monitor SIT protein levels during synchronized progression through the cell cycle. Peaks in SIT protein levels correlated with active periods of silica incorporation into cell wall substructures. Quantitative real-time PCR on each of the three distinct SIT genes (TpSIT1, TpSIT2, and TpSIT3) showed that mRNA levels for the most highly expressed SIT genes peaked during the S phase of the cell cycle, a period prior to maximal silicon uptake and during which cell wall silicification does not occur. Variations in protein and mRNA levels did not correlate, suggesting that a significant regulatory step of SITs is at the translational or posttranslational level. Surge uptake rates also did not correlate with SIT protein levels, suggesting that SIT activity is internally controlled by the rate of silica incorporation. This is the first study to characterize SIT mRNA and protein expression and cellular uptake kinetics during the course of the cell cycle and cell wall synthesis, and it provides novel insight into SIT regulation.

Functional Domains and DNA-binding Sequences of RFLAT-1/KLF13, a Krüppel-like Transcription Factor of Activated T Lymphocytes

RFLAT-1/KLF13, a member of the Krüppel-like family of transcription factors, was identified as a transcription factor expressed 3–5 days after T lymphocyte activation. It binds to the promoter of the chemokine gene RANTES (regulated on activation normal T cell expressed and secreted) and regulates its “late” expression in activated T-cells. In this study, a series of experiments to define the functional domains of RFLAT-1/KLF13 were undertaken to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this factor. Using the GAL4 fusion system, distinct transcriptional activation and repression domains were identified. The RFLAT-1 minimum activation domain is localized to amino acids 1–35, whereas the repression domain resides in amino acids 67–168. Deletion analysis on the RFLAT-1 protein further supports these domain functions. The RFLAT-1 activation domain is similar to that of its closest family member, basic transcription element-binding protein 1. This domain is highly hydrophobic, and site-directed mutagenesis demonstrated that both negatively charged and hydrophobic residues are important for transactivation. The nuclear localization signal of RFLAT-1 was also identified using the RFLAT-1/green fluorescence protein fusion approach. RFLAT-1 contains two potent, independent nuclear localization signals; one is immediately upstream of the zinc finger DNA-binding domain, and the other is within the zinc fingers. Using mutational analysis, we also determined that the critical binding sequence of RFLAT-1 is CTCCC. The intact CTCCC box on the RANTESpromoter is necessary for RFLAT-1-mediated RANTEStranscription and is also required for the synergy between RFLAT-1 and NF-κB proteins.

The multiple fates of sinking particles in the North Atlantic Ocean

The direct respiration of sinking organic matter by attached bacteria is often invoked as the dominant sink for settling particles in the mesopelagic ocean. However, other processes, such as enzymatic solubilization and mechanical disaggregation, also contribute to particle flux attenuation by transferring organic matter to the water column. Here we use observations from the North Atlantic Ocean, coupled to sensitivity analyses of a simple model, to assess the relative importance of particle-attached microbial respiration compared to the other processes that can degrade sinking particles. The observed carbon fluxes, bacterial production rates, and respiration by water column and particle-attached microbial communities each spanned more than an order of magnitude. Rates of substrate-specific respiration on sinking particle material ranged from 0.007 ± 0.003 to 0.173 ± 0.105 day−1. A comparison of these substrate-specific respiration rates with model results suggested sinking particle material was transferred to the water column by various biological and mechanical processes nearly 3.5 times as fast as it was directly respired. This finding, coupled with strong metabolic demand imposed by measurements of water column respiration (729.3 ± 266.0 mg C m−2 d−1, on average, over the 50 to 150 m depth interval), suggested a large fraction of the organic matter evolved from sinking particles ultimately met its fate through subsequent remineralization in the water column. At three sites, we also measured very low bacterial growth efficiencies and large discrepancies between depth-integrated mesopelagic respiration and carbon inputs.

Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light availability

Significance Diatoms are unicellular eukaryotic phytoplankton responsible for nearly one-half of total marine primary productivity. We identified a plastid-targeted protein in the coastal diatom Thalassiosira pseudonana (TpDSP1) that enhances growth during iron limitation under low light. Clone lines overexpressing TpDSP1 had lower quantum requirements for growth, increased levels of photosynthetic and carbon fixation proteins, and increased cyclic electron flow around photosystem I, an energy-producing pathway with a heretofore unappreciated role in diatoms. At the same time, clones growing under replete conditions had markedly reduced growth and photosynthetic rates under high light, suggesting that, although TpDSP1 confers a competitive advantage under iron limitation, cells walk an ecological tightrope through the regulation of this protein. Diatoms, unicellular phytoplankton that account for ∼40% of marine primary productivity, often dominate coastal and open-ocean upwelling zones. Limitation of growth and productivity by iron at low light is attributed to an elevated cellular Fe requirement for the synthesis of Fe-rich photosynthetic proteins. In the dynamic coastal environment, Fe concentrations and daily surface irradiance levels can vary by two to three orders of magnitude on short spatial and temporal scales. Although genome-wide studies are beginning to provide insight into the molecular mechanisms used by diatoms to rapidly respond to such fluxes, their functional role in mediating the Fe stress response remains uncharacterized. Here, we show, using reverse genetics, that a death-specific protein (DSP; previously named for its apparent association with cell death) in the coastal diatom Thalassiosira pseudonana (TpDSP1) localizes to the plastid and enhances growth during acute Fe limitation at subsaturating light by increasing the photosynthetic efficiency of carbon fixation. Clone lines overexpressing TpDSP1 had a lower quantum requirement for growth, increased levels of photosynthetic and carbon fixation proteins, and increased cyclic electron flow around photosystem I. Cyclic electron flow is an ATP-producing pathway essential in higher plants and chlorophytes with a heretofore unappreciated role in diatoms. However, cells under replete conditions were characterized as having markedly reduced growth and photosynthetic rates at saturating light, thereby constraining the benefits afforded by overexpression. Widespread distribution of DSP-like sequences in environmental metagenomic and metatranscriptomic datasets highlights the presence and relevance of this protein in natural phytoplankton populations in diverse oceanic regimes.

Expression, purification, and reconstitution of a diatom silicon transporter

The synthesis and manipulation of silicon materials on the nanoscale are core themes in nanotechnology research. Inspiration is increasingly being taken from the natural world because the biological mineralization of silicon results in precisely controlled, complex silica structures with dimensions from the millimeter to the nanometer. One fascinating example of silicon biomineralization occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall. To harvest silicon from the environment, diatoms have developed a unique family of integral membrane proteins that bind to a soluble form of silica, silicic acid, and transport it across the cell membrane to the cell interior. These are the first proteins shown to directly interact with silicon, but the current understanding of these specific silicon transport proteins is limited by the lack of in vitro studies of structure and function. We report here the recombinant expression, purification, and reconstitution of a silicon transporter from the model diatom Thalassiosira pseudonana. After using GFP fusions to optimize expression and purification protocols, a His(10)-tagged construct was expressed in Saccharomyces cerevisiae, solubilized in the detergent Fos-choline-12, and purified by affinity chromatography. Size-exclusion chromatography and particle sizing by dynamic light scattering showed that the protein was purified as a homotetramer, although nonspecific oligomerization occurred at high protein concentrations. Circular dichroism measurements confirmed sequence-based predictions that silicon transporters are α-helical membrane proteins. Silicic acid transport could be established in reconstituted proteoliposomes, and silicon uptake was found to be dependent upon an applied sodium gradient. Transport data across different substrate concentrations were best fit to the sigmoidal Hill equation, with a K(0.5) of 19.4 ± 1.3 μM and a cooperativity coefficient of 1.6. Sodium binding was noncooperative with a K(m)(app) of 1.7 ± 1.0 mM, suggesting a transport silicic acid:Na(+) stoichiometry of 2:1. These results provide the basis for a full understanding of both silicon transport in the diatom and protein-silicon interactions in general.

Diatom genomes come of age

The results of two published genome sequences from marine diatoms provide basic insights into how these remarkable organisms evolved to become one of the most successful groups of eukaryotic algae in the contemporary ocean.

When to say when: can excessive drinking explain silicon uptake in diatoms?

Diatoms are the single most important drivers of the oceanic silicon biogeochemical cycle. Due to their considerable promise in nanotechnology, there is tremendous interest in understanding the mechanism by which they produce their intricately and ornately decorated silica‐based cell wall. Although specific proteins have been implicated in some of the key steps of silicification, the exact mechanisms are poorly understood.

Coccolithovirus facilitation of carbon export in the North Atlantic

Marine phytoplankton account for approximately half of global primary productivity1, making their fate an important driver of the marine carbon cycle. Viruses are thought to recycle more than one-quarter of oceanic photosynthetically fixed organic carbon2, which can stimulate nutrient regeneration, primary production and upper ocean respiration2 via lytic infection and the ‘virus shunt’. Ultimately, this limits the trophic transfer of carbon and energy to both higher food webs and the deep ocean2. Using imagery taken by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Aqua satellite, along with a suite of diagnostic lipid- and gene-based molecular biomarkers, in situ optical sensors and sediment traps, we show that Coccolithovirus infections of mesoscale (~100 km) Emiliania huxleyi blooms in the North Atlantic are coupled with particle aggregation, high zooplankton grazing and greater downward vertical fluxes of both particulate organic and particulate inorganic carbon from the upper mixed layer. Our analyses captured blooms in different phases of infection (early, late and post) and revealed the highest export flux in ‘early-infected blooms’ with sinking particles being disproportionately enriched with infected cells and subsequently remineralized at depth in the mesopelagic. Our findings reveal viral infection as a previously unrecognized ecosystem process enhancing biological pump efficiency.Using a combination of remote-sensing technologies, lipidomics and gene-based biomarkers, the authors demonstrate a coupling between viral infection of an Emiliania huxleyi bloom and the export of organic and inorganic carbon from the photic zone.