RESUMO
Ecophysiological stress and the grazing of diatoms are known to elicit the production of chemical defense compounds called oxylipins, which are toxic to a wide range of marine organisms. Here we show that (1) the viral infection and lysis of diatoms resulted in oxylipin production; (2) the suite of compounds produced depended on the diatom host and the infecting virus; and (3) the virus-mediated oxylipidome was distinct, in both magnitude and diversity, from oxylipins produced due to stress associated with the growth phase. We used high-resolution accurate-mass mass spectrometry to observe changes in the dissolved lipidome of diatom cells infected with viruses over 3 to 4 days, compared to diatom cells in exponential, stationary, and decline phases of growth. Three host virus pairs were used as model systems: Chaetoceros tenuissimus infected with CtenDNAV; C. tenuissimus infected with CtenRNAV; and Chaetoceros socialis infected with CsfrRNAV. Several of the compounds that were significantly overproduced during viral infection are known to decrease the reproductive success of copepods and interfere with microzooplankton grazing. Specifically, oxylipins associated with allelopathy towards zooplankton from the 6-, 9-, 11-, and 15-lipogenase (LOX) pathways were significantly more abundant during viral lysis. 9-hydroperoxy hexadecatetraenoic acid was identified as the strongest biomarker for the infection of Chaetoceros diatoms. C. tenuissimus produced longer, more oxidized oxylipins when lysed by CtenRNAV compared to CtenDNAV. However, CtenDNAV caused a more statistically significant response in the lipidome, producing more oxylipins from known diatom LOX pathways than CtenRNAV. A smaller set of compounds was significantly more abundant in stationary and declining C. tenuissimus and C. socialis controls. Two allelopathic oxylipins in the 15-LOX pathway and essential fatty acids, arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were more abundant in the stationary phase than during the lysis of C. socialis. The host-virus pair comparisons underscore the species-level differences in oxylipin production and the value of screening more host-virus systems. We propose that the viral infection of diatoms elicits chemical defense via oxylipins which deters grazing with downstream trophic and biogeochemical effects.
Assuntos
Alelopatia , Diatomáceas , Oxilipinas , Oxilipinas/metabolismo , Animais , Organismos Aquáticos , ZooplânctonRESUMO
Algal viruses are important contributors to carbon cycling, recycling nutrients and organic material through host lysis. Although viral infection has been described as a primary mechanism of phytoplankton mortality, little is known about host defense responses. We show that viral infection of the bloom-forming, planktonic diatom Chaetoceros socialis induces the mass formation of resting spores, a heavily silicified life cycle stage associated with carbon export due to rapid sinking. Although viral RNA was detected within spores, mature virions were not observed. 'Infected' spores were capable of germinating, but did not propagate or transmit infectious viruses. These results demonstrate that diatom spore formation is an effective defense strategy against viral-mediated mortality. They provide a possible mechanistic link between viral infection, bloom termination, and mass carbon export events and highlight an unappreciated role of viruses in regulating diatom life cycle transitions and ecological success.
Assuntos
Diatomáceas , Vírus , Vírus de DNA , Mecanismos de Defesa , FitoplânctonRESUMO
Diatoms are prominent eukaryotic phytoplankton despite being limited by the micronutrient iron in vast expanses of the ocean. As iron inputs are often sporadic, diatoms have evolved mechanisms such as the ability to store iron that enable them to bloom when iron is resupplied and then persist when low iron levels are reinstated. Two iron storage mechanisms have been previously described: the protein ferritin and vacuolar storage. To investigate the ecological role of these mechanisms among diatoms, iron addition and removal incubations were conducted using natural phytoplankton communities from varying iron environments. We show that among the predominant diatoms, Pseudo-nitzschia were favored by iron removal and displayed unique ferritin expression consistent with a long-term storage function. Meanwhile, Chaetoceros and Thalassiosira gene expression aligned with vacuolar storage mechanisms. Pseudo-nitzschia also showed exceptionally high iron storage under steady-state high and low iron conditions, as well as following iron resupply to iron-limited cells. We propose that bloom-forming diatoms use different iron storage mechanisms and that ferritin utilization may provide an advantage in areas of prolonged iron limitation with pulsed iron inputs. As iron distributions and availability change, this speculated ferritin-linked advantage may result in shifts in diatom community composition that can alter marine ecosystems and biogeochemical cycles.
Assuntos
Diatomáceas/metabolismo , Ferro/metabolismo , Diatomáceas/classificação , Diatomáceas/crescimento & desenvolvimento , Ecossistema , Ferritinas/metabolismo , Fitoplâncton/crescimento & desenvolvimento , Fitoplâncton/metabolismoRESUMO
Viruses that infect photoautotrophs have a fundamental relationship with light, given the need for host resources. We investigated the role of light on Coccolithovirus (EhV) infection of the globally distributed coccolithophore, Emiliania huxleyi. Light was required for EhV adsorption, and viral production was highest when host cultures were maintained in continuous light or at irradiance levels of 150-300 µmol m-2 s-1 . During the early stages of infection, photosynthetic electron transport remained high, while RuBisCO expression decreased concomitant with an induction of the pentose phosphate pathway, the primary source of de novo nucleotides. A mathematical model developed and fitted to the laboratory data supported the hypothesis that EhV replication was controlled by a trade-off between host nucleotide recycling and de novo synthesis, and that photoperiod and photon flux could toggle this switch. Laboratory results supported field observations that light was the most robust driver of EhV replication within E. huxleyi populations collected across a 2000 nautical mile transect in the North Atlantic. Collectively, these findings demonstrate that light can drive host-virus interactions through a mechanistic interplay between host metabolic processes, which serve to structure infection and phytoplankton mortality in the upper ocean.
Assuntos
Haptófitas/efeitos da radiação , Haptófitas/virologia , Interações Hospedeiro-Patógeno/efeitos da radiação , Luz , Phycodnaviridae/fisiologia , Adsorção , Haptófitas/crescimento & desenvolvimento , NADP/metabolismo , Nucleotídeos/biossíntese , Via de Pentose Fosfato/efeitos da radiação , Fotoperíodo , Fotossíntese/efeitos da radiaçãoRESUMO
While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1 and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.
Assuntos
Interações entre Hospedeiro e Microrganismos , Marcação por Isótopo , Espectrometria de Massa de Íon Secundário , Vírus , Microbiologia da Água , Aminoácidos/análise , Fluorescência , Haptófitas , Synechococcus , Fenômenos Fisiológicos ViraisRESUMO
Frequent blooms of phytoplankton occur in coastal upwelling zones creating hotspots of biological productivity in the ocean. As cold, nutrient-rich water is brought up to sunlit layers from depth, phytoplankton are also transported upwards to seed surface blooms that are often dominated by diatoms. The physiological response of phytoplankton to this process, commonly referred to as shift-up, is characterized by increases in nitrate assimilation and rapid growth rates. To examine the molecular underpinnings behind this phenomenon, metatranscriptomics was applied to a simulated upwelling experiment using natural phytoplankton communities from the California Upwelling Zone. An increase in diatom growth following 5 days of incubation was attributed to the genera Chaetoceros and Pseudo-nitzschia. Here, we show that certain bloom-forming diatoms exhibit a distinct transcriptional response that coordinates shift-up where diatoms exhibited the greatest transcriptional change following upwelling; however, comparison of co-expressed genes exposed overrepresentation of distinct sets within each of the dominant phytoplankton groups. The analysis revealed that diatoms frontload genes involved in nitrogen assimilation likely in order to outcompete other groups for available nitrogen during upwelling events. We speculate that the evolutionary success of diatoms may be due, in part, to this proactive response to frequently encountered changes in their environment.
Assuntos
Diatomáceas/classificação , Diatomáceas/genética , Fitoplâncton/classificação , Fitoplâncton/genética , Evolução Biológica , California , Diatomáceas/metabolismo , Ecossistema , Expressão Gênica , Fitoplâncton/metabolismoRESUMO
Diatoms, unicellular phytoplankton that account for â¼40% 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.
Assuntos
Diatomáceas/genética , Ferro/análise , Luz , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas/genética , Biofísica , Carbono/análise , Clonagem Molecular , Diatomáceas/crescimento & desenvolvimento , Immunoblotting , Microscopia de Fluorescência , Nitrogênio/análise , Fotossíntese/genética , Proteínas/fisiologiaRESUMO
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.
Assuntos
Diatomáceas/química , Proteínas de Membrana Transportadoras/metabolismo , Ácido Silícico/metabolismo , Transporte Biológico , Parede Celular/química , Detergentes/farmacologia , Proteínas de Membrana Transportadoras/isolamento & purificação , Nanotecnologia/métodos , Proteolipídeos/efeitos dos fármacos , Proteolipídeos/metabolismo , Proteínas Recombinantes/isolamento & purificação , Silício/metabolismo , SolubilidadeRESUMO
Low iron (Fe) availability critically limits diatom distribution and productivity in vast regions of the modern ocean, such as open-ocean, high nutrient low chlorophyll areas and coastal regimes characterized as Fe limitation 'mosaics'. While unique strategies of Fe uptake and storage confer competitive advantages to pennate diatoms, the molecular determinants of low Fe acclimation are largely unknown in centric diatoms. We combined genome-wide and targeted comparative transcriptomic analysis with diagnostic biochemistry and in vivo cell staining as a platform to identify the suite of genes involved in acclimation to Fe and associated oxidative stress in Thalassiosira pseudonana. A total of 1312 genes, nearly 12% of the total genome content, responded to Fe starvation in growing cells characterized by low photosynthetic efficiency and enhanced oxidative stress, caspase activity and metacaspase expression. While 82% of the most highly upregulated genes were also represented in EST libraries derived from diverse diatoms grown under various stress conditions (e.g. silicon, CO(2) and nitrogen limitation), our analysis suggests that T. pseudonana mounts a unique molecular response to Fe starvation that includes a number of genes distinct from those of the model pennate diatom, Phaeodactylum tricornutum, which diverged ~90 million years ago. Homologues to ~50% of the upregulated genes were also identified in a metatranscriptome of eukaryotic phytoplankton communities from a chronically Fe-limited region in the Northeast Pacific. Furthermore, we provide experimental evidence that a subset of putative death-related genes participate in the cellular acclimation to low Fe and associated oxidative damage, suggesting that they co-evolved with other metabolic pathways and play adaptive roles in the success of diatoms.
Assuntos
Aclimatação/genética , Diatomáceas/fisiologia , Ferro/metabolismo , Estresse Oxidativo/genética , Transcriptoma , Morte Celular , Diatomáceas/genética , Evolução Molecular , Genoma , Nitrogênio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fotossíntese , Espécies Reativas de Oxigênio/metabolismo , Silício/metabolismoRESUMO
In the California Current Ecosystem, upwelled water low in dissolved iron (Fe) can limit phytoplankton growth, altering the elemental stoichiometry of the particulate matter and dissolved macronutrients. Iron-limited diatoms can increase biogenic silica (bSi) content >2-fold relative to that of particulate organic carbon (C) and nitrogen (N), which has implications for carbon export efficiency given the ballasted nature of the silica-based diatom cell wall. Understanding the molecular and physiological drivers of this altered cellular stoichiometry would foster a predictive understanding of how low Fe affects diatom carbon export. In an artificial upwelling experiment, water from 96 m depth was incubated shipboard and left untreated or amended with dissolved Fe or the Fe-binding siderophore desferrioxamine-B (+DFB) to induce Fe-limitation. After 120 h, diatoms dominated the communities in all treatments and displayed hallmark signatures of Fe-limitation in the +DFB treatment, including elevated particulate Si:C and Si:N ratios. Single-cell, taxon-resolved measurements revealed no increase in bSi content during Fe-limitation despite higher transcript abundance of silicon transporters and silicanin-1. Based on these findings we posit that the observed increase in bSi relative to C and N was primarily due to reductions in C fixation and N assimilation, driven by lower transcript expression of key Fe-dependent genes.
RESUMO
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.Silicon transporters, identified in both diatoms and silicoflagellates, are hypothesized to mediate silicon uptake. Recently, macropinocytosis, the non-specific engulfment of extracellular fluid, was proposed as a more energetically favorable uptake mechanism, which can also explain the long-observed effect of salinity on frustule morphology. We explore the bioenergetic, membrane recycling, and vacuolar volume requirements that must be satisfied for pinocytosis-mediated silicon uptake. These calculated requirements contrast starkly with existing data on diatom physiology, uptake kinetics, growth, and ultrastructure, leading us to conclude that pinocytosis cannot be the primary mechanism of silicon uptake.
Assuntos
Diatomáceas/metabolismo , Silício/metabolismo , Água/metabolismo , Membrana Celular/metabolismo , Metabolismo Energético , CinéticaRESUMO
A new study reveals that phosphorus-limited diatoms employ a rapid calcium-based signaling pathway upon phosphorus resupply. This response leads to enhanced nitrogen uptake and assimilation, setting the stage for recovery from nutrient deprivation.
Assuntos
Diatomáceas , Nitrogênio , Nutrientes , FósforoRESUMO
The blooming cosmopolitan coccolithophore Emiliania huxleyi and its viruses (EhVs) are a model for density-dependent virulent dynamics. EhVs commonly exhibit rapid viral reproduction and drive host death in high-density laboratory cultures and mesocosms that simulate blooms. Here we show that this system exhibits physiology-dependent temperate dynamics at environmentally relevant E. huxleyi host densities rather than virulent dynamics, with viruses switching from a long-term non-lethal temperate phase in healthy hosts to a lethal lytic stage as host cells become physiologically stressed. Using this system as a model for temperate infection dynamics, we present a template to diagnose temperate infection in other virus-host systems by integrating experimental, theoretical, and environmental approaches. Finding temperate dynamics in such an established virulent host-virus model system indicates that temperateness may be more pervasive than previously considered, and that the role of viruses in bloom formation and decline may be governed by host physiology rather than by host-virus densities.
Assuntos
Haptófitas/virologia , Vírus de Plantas/fisiologia , Vírus de Plantas/patogenicidade , Haptófitas/fisiologia , Interações Hospedeiro-Patógeno , Modelos Biológicos , VirulênciaRESUMO
Diatoms are among the most globally distributed and ecologically successful organisms in the modern ocean, contributing upwards of 40% of total marine primary productivity1,2. By converting dissolved silicon into biogenic silica, and photosynthetically fixing carbon dioxide into particulate organic carbon, diatoms effectively couple the silicon (Si) and carbon cycles and ballast substantial vertical flux of carbon out of the euphotic zone into the mesopelagic and deep ocean3-5. Viruses are key players in ocean biogeochemical cycles6,7, yet little is known about how viral infection specifically impacts diatom populations. Here, we show that Si limitation facilitates virus infection and mortality in diatoms in the highly productive coastal waters of the California Current Ecosystem. Using metatranscriptomic analysis of cell-associated diatom viruses and targeted quantification of extracellular viruses, we found a link between Si stress and the early, active and lytic stages of viral infection. This relationship was also observed in cultures of the bloom-forming diatom Chaetoceros tenuissimus, where Si stress accelerated virus-induced mortality. Together, these findings contextualize viruses within the ecophysiological framework of Si availability and diatom-mediated biogeochemical cycling.
Assuntos
Diatomáceas/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Silício/metabolismo , Vírus/patogenicidade , Biodegradação Ambiental , California , Carbono/metabolismo , Dióxido de Carbono , Diatomáceas/metabolismo , Diatomáceas/virologia , Metagenômica , Análise de Sequência de RNA , Vírus/classificação , Vírus/genéticaRESUMO
Marine phytoplankton account for approximately half of global primary productivity 1 , 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 carbon 2 , which can stimulate nutrient regeneration, primary production and upper ocean respiration 2 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 ocean 2 . 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.
Assuntos
Carbono/metabolismo , Haptófitas/virologia , Phycodnaviridae/fisiologia , Ciclo do Carbono , Cadeia Alimentar , Haptófitas/fisiologia , Oceanos e Mares , Fitoplâncton/fisiologia , Fitoplâncton/virologia , Tecnologia de Sensoriamento Remoto , Imagens de Satélites , Água do Mar/virologiaRESUMO
A major component of the diatom cell wall is silica, derived from silicon taken up from the environment. Due to limiting environmental concentrations of silicon, and a substantial requirement during cell wall synthesis, diatoms must transport silicon into the cell against a steep concentration gradient. This is accomplished through the silicic acid transporters (SITs). The SITs were first identified in the marine pennate diatom Cylindrotheca fusiformis. Five distinct SITs were found and have been classified as a novel family of transporters. This review covers our current understanding of silicon transport in diatoms with a focus on the SITs. Approaches for in-depth functional characterization of the SITs are discussed, including (1) isolating SITs from evolutionarily distant diatom species to identify conserved amino acids that may be important for function, (2) developing expression systems to assay the function of selected SITs, and (3) determining the cellular location and membrane topology of the C. fusiformis SITs to further clarify their roles in diatom silicon metabolism. Because of the specificity of interaction between the SITs and silicon, and the ability of the SITs to transport silicic acid across lipid bilayers, the SITs may have applications in nanotechnology.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Diatomáceas/metabolismo , Ácido Silícico/metabolismo , Dióxido de Silício/metabolismo , Silício/metabolismo , Sequência de Aminoácidos , Transporte Biológico Ativo/fisiologia , Biomimética/métodos , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
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 (68)Ge(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.
Assuntos
Diatomáceas/metabolismo , Modelos Biológicos , Ácido Silícico/metabolismo , Parede Celular/metabolismo , Diatomáceas/crescimento & desenvolvimento , Difusão , Germânio/metabolismo , Espaço Intracelular/metabolismo , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Radioisótopos/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Diatomáceas/genética , Genoma , Evolução Biológica , DNA de Algas/genéticaRESUMO
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.
Assuntos
Proteínas de Algas/metabolismo , Transporte Biológico , Diatomáceas/química , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Fase S , Silício/metabolismo , Sequência de Aminoácidos , Parede Celular/química , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
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 RANTES promoter is necessary for RFLAT-1-mediated RANTES transcription and is also required for the synergy between RFLAT-1 and NF-kappaB proteins.