Project description:Diatoms perform about 20% of photosynthesis on earth, play a crucial role in forming the base of the marine food web, and sequester anthropogenic carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the organic micronutrient cobalamin (vitamin B12). Cobalamin affects phytoplankton growth rate and biomass yield, though the consequences of cobalamin limitation are not well understood on the biochemical level. In a controlled laboratory setting, we grew two diatoms, Thalassiosira pseudonana and Navicula pelliculosa, under cobalamin limited and replete conditions to elucidate changes in metabolite pools under cobalamin limitation. Using targeted and untargeted metabolomics, we show that the cobalamin-dependent T. pseudonana experienced a metabolic cascade under cobalamin limitation that a ects the central methionine cycle, the transsulfuration pathway, and the composition of its osmolyte pools. N. pelliculosa, a cobalamin-independent diatom, also showed evidence for changes in the methionine cycle when grown without cobalamin, though to a lesser extent than what we observed in T. pseudonana. In both diatoms the concentration of 5'-methylthioadenosine decreased when limited for cobalamin, suggesting a disruption in the diatoms' polyamine biosynthesis. Furthermore, two acylcarnitines and adenosyl cobalamin accumulated in T. pseudonana under cobalamin limitation, suggesting the use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin starvation may have consequences for microbial interactions that are based on metabolite production by phytoplankton. Navicula pelliculosa protocols and data are reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS708' rel='noopener noreferrer' target='_blank'>MTBLS708</a>. Thalassiosira pseudonana protocols and data are reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS703' rel='noopener noreferrer' target='_blank'>MTBLS703</a>.

Project description:Diatoms perform about 20% of photosynthesis on earth, play a crucial role in forming the base of the marine food web, and sequester anthropogenic carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the organic micronutrient cobalamin (vitamin B12). Cobalamin affects phytoplankton growth rate and biomass yield, though the consequences of cobalamin limitation are not well understood on the biochemical level. In a controlled laboratory setting, we grew two diatoms, Thalassiosira pseudonana and Navicula pelliculosa, under cobalamin limited and replete conditions to elucidate changes in metabolite pools under cobalamin limitation. Using targeted and untargeted metabolomics, we show that the cobalamin-dependent T. pseudonana experienced a metabolic cascade under cobalamin limitation that a ects the central methionine cycle, the transsulfuration pathway, and the composition of its osmolyte pools. N. pelliculosa, a cobalamin-independent diatom, also showed evidence for changes in the methionine cycle when grown without cobalamin, though to a lesser extent than what we observed in T. pseudonana. In both diatoms the concentration of 5'-methylthioadenosine decreased when limited for cobalamin, suggesting a disruption in the diatoms' polyamine biosynthesis. Furthermore, two acylcarnitines and adenosyl cobalamin accumulated in T. pseudonana under cobalamin limitation, suggesting the use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin starvation may have consequences for microbial interactions that are based on metabolite production by phytoplankton. Thalassiosira pseudonana protocols and data are reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS703' rel='noopener noreferrer' target='_blank'>MTBLS703</a>. Navicula pelliculosa protocols and data are reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS708' rel='noopener noreferrer' target='_blank'>MTBLS708</a>.

Project description:Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three marine diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. Less than 5% of orthologous genes, shared across the three diatoms, displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling. Transcriptomes were collected for diatom cultures harvested at the onset of stationary phase in low nitrate media (55 M-NM-<M NaNO3, 212 M-NM-<M Na2SiO3, 72.4 M-NM-<M NaH2PO4) or during mid-exponential growth in nutrient-replete media (882 M-NM-<M NaNO3, 106 M-NM-<M Na2SiO3, 36.2 M-NM-<M NaH2PO4) in artificial seawater, maintaining three biological replicates per condition and per diatom (N=18). The SOLiD sequencer (version 4) was used to generate the transcriptomes and the SEAStAR software package was used to process the SOLiD reads and to calculate gene counts. Pooled counts for the nitrate-limited treatment were normalized to pooled counts for the nutrient-replete M-bM-^@M-^\controlM-bM-^@M-^] treatment to generate log fold changes in gene transcription using the R software package edgeR from Bioconductor.

Project description:Diatoms are responsible for ~40% of marine primary productivity1, fueling the oceanic carbon cycle and contributing to natural carbon sequestration in the deep ocean2. Diatoms rely on energetically expensive carbon concentrating mechanisms (CCMs) to fix carbon efficiently at modern levels of CO23–5. How diatoms may respond over the short and long-term to rising atmospheric CO2 remains an open question. Here we use nitrate-limited chemostats to show that the model diatom Thalassiosira pseudonana rapidly responds to increasing CO2 by differentially expressing gene clusters that regulate transcription and chromosome folding and subsequently reduces transcription of photosynthesis and respiration gene clusters under steady-state elevated CO2. These results suggest that exposure to elevated CO2 first causes a shift in regulation and then a metabolic rearrangement. Genes in one CO2-responsive cluster included CCM and photorespiration genes that share a putative cyclic-AMP responsive cis-regulatory sequence, implying these genes are co-regulated in response to CO2 with cAMP as an intermediate messenger. We verified cAMP-induced down-regulation of CCM gene ?-CA3 in nutrient-replete diatom cultures by inhibiting the hydrolysis of cAMP. These results indicate an important role for cAMP in down-regulating CCM and photorespiration genes under elevated CO2 and provide insights into mechanisms of diatom acclimation in response to climate change. In steady-state experiments: axenic T. pseudonana cells in four biological replicates (duplicate chemostats x 2 experimental runs) were acclimated to nitrate-limitation at 70% (1.5 day-1) of max growth rate for >10 days (>15 generations) under continuous light of 80 µmol photons·m?2·s?1. Cell biomass was maintained at ~2 x 105 cells·mL?1 by 10 ?M nitrate and carbonate chemistry stabilized to 300, 475 or 800 ?atm CO2, verified by pH and DIC measurements. Transition samples and carbonate chemistry were collected daily from chemostat cultures as CO2 levels were increased from ~300-800 ?atm at a rate ? 0.2 ?atm·min?1 over four consecutive days (6 generations) after pre-acclimation to 300 ?atm CO2 and nitrate-limitation.

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MetaboLights MTBLS708 - GNPS Metabolic consequences of cobalamin scarcity in diatoms as revealed through metabolomics (Navicula pelliculosa study)

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