Metabolic analysis of adaptation to short-term changes in culture conditions of the marine diatom Thalassiosira pseudonana.
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ABSTRACT:
SUBMITTER: Bromke MA
PROVIDER: S-EPMC3682967 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the modern ocean, phytoplankton maintain extremely high primary production/biomass ratios, indicating that they bloom, die, and are replaced weekly. The molecular mechanisms regulating cellular mortality and turnover are largely unknown, even though they effectively short-circuit carbon export to the deep ocean and channel primary productivity to microbial food webs. Here, we present morphological, biochemical, and molecular evidence of caspase-mediated, autocatalytic programmed cell death (PCD) in the diatom Thalassiosira pseudonana in response to iron starvation. Transmission electron microscopy revealed internal degradation of nuclear, chloroplastic, and mitochondrial organelles, all while the plasma membranes remained intact. Cellular degradation was concomitant with dramatic decreases in photosynthetic efficiency, externalization of phosphatidylserine, and significantly elevated caspase-specific activity, with the addition of a broad-spectrum caspase inhibitor rescuing cells from death. A search of the T. pseudonana genome identified six distinct putative metacaspases containing a conserved caspase domain structure. Quantitative reverse transcription-PCR and Western blot analysis revealed differential gene and protein expression of T. pseudonana metacaspases, some of which correlated with physiological stress and caspase activity. Taken together with the recent discovery of the metacaspase-mediated viral infection of phytoplankton (K. D. Bidle, L. Haramaty, J. Barcelos-Ramos, and P. G. Falkowski, Proc. Natl. Acad. Sci. USA 104:6049-6054, 2007), our findings reveal a key role for metacaspases in the turnover of phytoplankton biomass in the oceans. Furthermore, given that Fe is required for photosynthetic electron transfer and is chronically limiting in a variety of oceanic systems, including high-nutrient low-chlorophyll regions, our findings provide a potential ecological context for PCD in these unicellular photoautotrophs.
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Project description:BackgroundCP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background.MethodsA combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12.ResultsHere, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions.ConclusionsThese findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.
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Project description:BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants ubiquitously distributed. They are generated by incomplete combustion of organic materials such as wood or fossil fuels. Due to their carcinogenic, mutagenic effects and to their wide distribution in the environment, these pollutants pose many concerns to researchers and regulators. In our laboratories we investigated the effect of benzo(a)pyrene (BaP) exposure in the marine diatom Thalassiosira pseudonana, which has become an important model organism in aquatic toxicology studies. RESULTS: In order to investigate the mechanism of action of PAHs, we exposed the diatoms for 24 h to 36.45 ?g/L of BaP which inhibits the growth by about 30%, and analysed the relative protein expression profile by a quantitative proteomics approach based on iTRAQ labels. The proteomics profile analysis showed that around 10% of the identified proteins were regulated and one fourth of them confirmed the gene expression changes seen by DNA microarray. Particularly interesting was the down regulation of the Silicon transporter 1 (SIT1), an enzyme that is responsible for the uptake of silicon from the media into the diatom cells. Regulation of SIT1 upon BaP treatment was also confirmed at the gene expression level. CONCLUSIONS: The potential use of the regulated proteins found in this study as early indicators of environmental exposure to PAHs is discussed. In particular, SIT1 is considered a promising biomarker and SIT1 expression changes were confirmed also when the diatoms were exposed to field samples, e.g. marine surface sediments contaminated by PAHs.
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Project description:Increased manufacture of TiO2 nanoproducts has caused concern about the potential toxicity of these products to the environment and in public health. Identification and confirmation of the presence of TiO2 nanoparticles derived from consumer products as opposed to industrial TiO2 NPs warrant examination in exploring the significance of their release and resultant impacts on the environment. To this end, we examined the significance of the release of these particles and their toxic effect on the marine diatom algae Thalassiosira pseudonana. Our results indicate that nano-TiO2 sunscreen and toothpaste exhibit more toxicity in comparison to industrial TiO2 and inhibited the growth of the marine diatom T. pseudonana. This inhibition was proportional to the exposure time and concentrations of nano-TiO2. Our findings indicate a significant effect, and therefore, further research is warranted in evaluation and assessment of the toxicity of modified nano-TiO2 derived from consumer products and their physicochemical properties.
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Project description:Formation of complex inorganic structures is widespread in nature. Diatoms create intricately patterned cell walls of inorganic silicon that are a biomimetic model for design and generation of three-dimensional silica nanostructures. To date, only relatively simple silica structures can be generated in vitro through manipulation of known diatom phosphoproteins (silaffins) and long-chain polyamines. Here, we report the use of genome-wide transcriptome analyses of the marine diatom Thalassiosira pseudonana to identify additional candidate gene products involved in the biological manipulation of silicon. Whole-genome oligonucleotide tiling arrays and tandem mass spectrometry identified transcripts for >8,000 genes, approximately 3,000 of which were not previously described and included noncoding and antisense RNAs. Gene-specific expression profiles detected a set of 75 genes induced only under low concentrations of silicon but not under low concentrations of nitrogen or iron, alkaline pH, or low temperatures. Most of these induced gene products were predicted to contain secretory signals and/or transmembrane domains but displayed no homology to known proteins. Over half of these genes were newly discovered, identified only through the use of tiling arrays. Unexpectedly, a common set of 84 genes were induced by both silicon and iron limitations, suggesting that biological manipulation of silicon may share pathways in common with iron or, alternatively, that iron may serve as a required cofactor for silicon processes. These results provide insights into the transcriptional and translational basis for the biological generation of elaborate silicon nanostructures by these ecologically important microbes.
| S-EPMC2234187 | biostudies-literature
Project description:BACKGROUND:Publication of the first diatom genome, that of Thalassiosira pseudonana, established it as a model species for experimental and genomic studies of diatoms. Virtually every ensuing study has treated T. pseudonana as a marine diatom, with genomic and experimental data valued for their insights into the ecology and evolution of diatoms in the world's oceans. RESULTS:The natural distribution of T. pseudonana spans both marine and fresh waters, and phylogenetic analyses of morphological and molecular datasets show that, 1) T. pseudonana marks an early divergence in a major freshwater radiation by diatoms, and 2) as a species, T. pseudonana is likely ancestrally freshwater. Marine strains therefore represent recent recolonizations of higher salinity habitats. In addition, the combination of a relatively nondescript form and a convoluted taxonomic history has introduced some confusion about the identity of T. pseudonana and, by extension, its phylogeny and ecology. We resolve these issues and use phylogenetic criteria to show that T. pseudonana is more appropriately classified by its original name, Cyclotella nana. Cyclotella contains a mix of marine and freshwater species and so more accurately conveys the complexities of the phylogenetic and natural histories of T. pseudonana. CONCLUSIONS:The multitude of physical barriers that likely must be overcome for diatoms to successfully colonize freshwaters suggests that the physiological traits of T. pseudonana, and the genes underlying those traits, might differ from those of strictly marine diatoms. The freshwater ancestry of T. pseudonana might therefore confound generalizations about the physiological and metabolic properties of marine diatoms. The freshwater component of T. pseudonana's history merits careful consideration in the interpretation of experimental data collected for this important model species.
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Project description:In the following article an electron/ion microscopy study will be presented which investigates the uptake of silver nanoparticles (AgNPs) by the marine diatom Thalassiosira pseudonana, a primary producer aquatic species. This organism has a characteristic silica exoskeleton that may represent a barrier for the uptake of some chemical pollutants, including nanoparticles (NPs), but that presents a technical challenge when attempting to use electron-microscopy (EM) methods to study NP uptake. Here we present a convenient method to detect the NPs interacting with the diatom cell. It is based on a fixation procedure involving critical point drying which, without prior slicing of the cell, allows its inspection using transmission electron microscopy. Employing a combination of electron and ion microscopy techniques to selectively cut the cell where the NPs were detected, we are able to demonstrate and visualize for the first time the presence of AgNPs inside the cell membrane.
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Project description:Diatoms are unicellular, photosynthetic, eukaryotic algae with a ubiquitous distribution in water environments and they play an important role in the carbon cycle. Molecular or morphological changes in these species under ecological stress conditions are expected to serve as early indicators of toxicity and can point to a global impact on the entire ecosystem. Thalassiosira pseudonana, a marine diatom and the first with a fully sequenced genome has been selected as an aquatic model organism for ecotoxicological studies using molecular tools. A customized DNA microarray containing probes for the available gene sequences has been developed and tested to analyze the effects of a common pollutant, benzo(a)pyrene (BaP), at a sub-lethal concentration. This approach in diatoms has helped to elucidate pathway/metabolic processes involved in the mode of action of this pollutant, including lipid metabolism, silicon metabolism and stress response. A dose-response of BaP on diatoms has been made and the effect of this compound on the expression of selected genes was assessed by quantitative real time-PCR. Up-regulation of the long-chain acyl-CoA synthetase and the anti-apoptotic transmembrane Bax inhibitor, as well as down-regulation of silicon transporter 1 and a heat shock factor was confirmed at lower concentrations of BaP, but not the heat-shock protein 20. The study has allowed the identification of molecular biomarkers to BaP to be later on integrated into environmental monitoring for water quality assessment.
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Project description:Bacteria-diatom interactions in the ocean are diverse but usually studied in static conditions, which limits our understanding of their importance in marine ecosystems and biogeochemical cycles. Here, we explored the dynamic interactions between an ubiquitous marine bacterium Alteromonas sp. and a diatom Thalassiosira pseudonana under different nutrient conditions. In oligotrophic conditions, minor shifts in nutrients qualitatively altered the interactions from mutualism during early exponential growth to weak parasitism during the late stationary phase. Organic matter-activated Alteromonas chemotactically swam toward, attached on, and dramatically broke down T. pseudonana cells, leading to an aggressive parasitic behavior with a 95% algicidal rate. Meanwhile, inorganic matter-activated T. pseudonana showed amensalism against Alteromonas, resulting in an ephemeral decrease of bacterial abundance by 27%. Interestingly, when both organics and inorganics were sufficient, Alteromonas suppressed diatom growth by inhibiting the cell division, while the surviving T. pseudonana restored proliferation with a significantly smaller cell size inconducive to bacterial attachment, demonstrating an intense competition. The results further indicated that the algicidal effect of Alteromonas was controlled by the cell-specific protease activity and the number of attached bacteria on the diatom cell surface, both of which were related to nutrient conditions. Since the nature and intensity of bacteria-diatom interaction depend on the composition and richness of nutrients, it mechanistically explains the tripartite relationship among bacterial proliferation, nutrient viability, and algal demise during blooms. The algicidal behavior of copiotrophs also potentially enhances the contribution of a microbial carbon pump to carbon sequestration in the ocean. IMPORTANCE As the major producers and consumers, phytoplankton and bacteria play central roles in marine ecosystems and their interactions show great ecological significance. Whether mutualistic or antagonistic, the interaction between certain phytoplankton and bacterial species is usually seen as a derivative of intrinsic physiological properties and rarely changes. This study demonstrated that the interactions between the ubiquitously co-occurring bacteria and diatom, Alteromonas and Thalassiosira pseudonana, varied with nutrient conditions. They overcame hardship together in oligotrophic seawater but showed antagonistic effects against each other under nutrient amendment. The contact-dependent algicidal behavior of Alteromonas based on protease activity solved the paradox among bacterial proliferation, nutrient viability, and algal demise haunting other known non-contact-dependent algicidal processes and might actually trigger the collapse of algal blooms in situ. The chemotactic and swarming movement of Alteromonas might also contribute greatly to the breakdown of "marine snow," which could redirect the carbon sequestration pathway in the ocean.
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Project description:Marine diatoms are important primary producers that thrive in diverse and dynamic environments. They do so, in theory, by sensing changing conditions and adapting their physiology accordingly. Using the model species Thalassiosira pseudonana, we conducted a detailed physiological and transcriptomic survey to measure the recurrent transcriptional changes that characterize typical diatom growth in batch culture. Roughly 40% of the transcriptome varied significantly and recurrently, reflecting large, reproducible cell-state transitions between four principal states: (i) "dawn," following 12 h of darkness; (ii) "dusk," following 12 h of light; (iii) exponential growth and nutrient repletion; and (iv) stationary phase and nutrient depletion. Increases in expression of thousands of genes at the end of the reoccurring dark periods (dawn), including those involved in photosynthesis (e.g., ribulose-1,5-bisphosphate carboxylase oxygenase genes rbcS and rbcL), imply large-scale anticipatory circadian mechanisms at the level of gene regulation. Repeated shifts in the transcript levels of hundreds of genes encoding sensory, signaling, and regulatory functions accompanied the four cell-state transitions, providing a preliminary map of the highly coordinated gene regulatory program under varying conditions. Several putative light sensing and signaling proteins were associated with recurrent diel transitions, suggesting that these genes may be involved in light-sensitive and circadian regulation of cell state. These results begin to explain, in comprehensive detail, how the diatom gene regulatory program operates under varying environmental conditions. Detailed knowledge of this dynamic molecular process will be invaluable for new hypothesis generation and the interpretation of genetic, environmental, and metatranscriptomic data from field studies.
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