Next generation sequencing for understading the role of nitrate reductase in remodeling intermediate metabolism in Phaeodactylum tricornutum
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ABSTRACT:
ORGANISM(S): Phaeodactylum tricornutum
SUBMITTER: Orly Levitan
PROVIDER: E-GEOD-67449 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We have done next generation sequencing of optically thin, exponentialy growing Phaeodactylum tricornutum cultures grown with and without nitrogen source to improve our undestanding of the pathways regulation under conditions that promote lipid accumulation (N-starvation). 3 biologically independent exponentially growing culture of Phaeodactylum tricornutum were pelleted and washed several times with N-free media. Each culture was devided to 2 replicates with initial cell concentration of 2X105 cells/mL, one with NaNO3 as nitrogen source and the other without any nitrogen source. The cuture were bubbled foro 48 hours and sampled for transcriptome together with other physiological parameters and lipid analysis.
2015-01-02 | E-GEOD-56346 | biostudies-arrayexpress
Project description:We have done next generation sequencing of optically thin, exponentialy growing WT Phaeodactylum tricornutum and our ~50% nitrate reductase knock-down cultures. Culture were grown with and without nitrogen source to improve our undestanding of the pathways regulation under conditions that promote lipid accumulation (N-starvation).
2016-03-31 | GSE67449 | GEO
Project description:Microarray analysis of Aspergillus niger under conditions with differing combinations of carbon source, nitrogen source, nitrogen concentration, and culture pH Fermentor cultures were grown in minimal medium (MM) at a constant temperature of 30 ± 0.5 ºC and with differing combinations of carbon source (either 277.5 mM glucose or 333.0 mM xylose), nitrogen source (NH4Cl or NaNO3) and nitrogen concentration (4x: 282.4 mM; 8x: 564.8 mM), and pH (pH4 or pH5) of the medium (M. Braaksma, A.K. Smilde, M.J. van der Werf, P.J. Punt, submitted for publication). At different time points samples were collected, quenched immediately in methanol at -45 ºC and centrifuged at -20 ºC to remove supernatant. Part of the biomass was frozen into liquid nitrogen and stored at -80 ºC for microarray analysis. For each of the 16 culture conditions one sample was selected for microarray analysis; samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion. In addition some technical duplicates were included. 20 samples were analyzed from 16 different fermentation conditions. The fermentation conditions were varied according to a full factorial design of four factors tested at two levels. From one fermentation two different time samples were analyzed, from another fermentation four samples, two technical replicates of two different time samples, were analyzed. Samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion.
2010-04-08 | E-GEOD-17329 | biostudies-arrayexpress
Project description:We have done next generation sequencing of optically thin, exponentialy growing Phaeodactylum tricornutum cultures grown with and without nitrogen source to improve our undestanding of the pathways regulation under conditions that promote lipid accumulation (N-starvation).
2015-01-02 | GSE56346 | GEO
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.
2014-03-25 | E-GEOD-56132 | biostudies-arrayexpress
Project description:An experiment was performed to determine the similarities on the RNA level between different conditions where cell division stops in the diatom Phaeodactylum tricornutum. Many of these conditions also increase the accumulation of lipids within the cell or impair photosynthesis. The different metabolic responses were evaluated and the dataset was mined for potential transcriptional regulators of these changes. The experimental setup was as follows: Cells from the pennate diatom Phaeodactylum tricornutum were grown in ESAW medium under continous fluorescent light at 21C in baffled shakeflasks. Exponentially growing cells were harvested by centrifugation and washed twice in 21gr/L NaCL to remove nutrients. Cells were subsequently resuspended in the five different media/conditions (control, darkness, no nitrate, no phosphate, nocodazole).
2015-12-07 | E-MTAB-4096 | biostudies-arrayexpress
Project description:The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is already well-characterized in heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles. This study reveals 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. Physiological characterization of a 6S RNA deletion strain (ÎssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation is accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding components of both photosystems, ATP synthase and the phycobilisomes were negatively affected in the ÎssaA mutant. In vivo pull-down studies of the RNA polymerase complex further indicate a promoting effect of 6S RNA on the recruitment of the cyanobacterial housekeeping sigma factor SigA, concurrently supporting dissociation of group II sigma factors during recovery from nitrogen starvation. According to these results, 6S RNA supports a rapid adaptation to changing nitrogen conditions by regulating the switch from group II sigma factors SigB / SigC to SigE / SigA dependent transcription. We performed microarray analysis of total RNA from wild-type and âssaA cultures that were starved for nitrogen for seven days and recovered over a period of 48 hours. Sampling time points were t1 = 1h +N, t2 = 4h +N and t3 = 22h +N after nitrogen recovery. Samples were taken in biological replicates.
2016-06-16 | E-GEOD-83387 | biostudies-arrayexpress
Project description:Unicellular cyanobacteria that do not fix nitrogen can survive prolonged periods of nitrogen starvation as bleached cells in a non-growing, dormant state. Upon re-addition of a usable nitrogen source, bleached cultures re-green within 48 hours and the cells return to vegetative growth. Here we investigated the process of resuscitation at the physiological and molecular level. Almost immediately upon nitrate addition, the cells initiate an amazingly organized resuscitation program: they first turn on respiration, gaining energy and activating the genes of the entire translational apparatus, genes for ATP synthesis and nitrate assimilation. Only after about 12 hours, the cells rebuild the photosynthetic apparatus and switch on photosynthesis. Analysis of the transcriptome in recovering cells shows a perfect match to the physiological processes and reveals a paramount dynamics of non-coding RNAs in awaking cells. This genetically encoded program ensures rapid colonization of habitats, in which nitrogen starvation imposes a recurring growth limitation. Synechocstis PCC 6803 WT cells were subjected to nitrogen limitation for 14d, then nitrogen was re-added to monitor recovery of the cells. Samples were taken before nitrogen depletion, after 14d of nitrogen depletion and 4h, 13h, 24h and 48h after nitrogen re-addition. Samples were taken in biological replicates for all timepoints besides 48h nitrogen recovery.
2016-06-15 | E-GEOD-83363 | biostudies-arrayexpress
Project description:To resolve the temporal sequence of phosphorylation events responding to nutritional and chemically induced up and downshifts of TORC1 signaling in S. cerevisiae wildtype, we performed quantitative MS phosphoproteomics upon nitrogen-quality up and downshifts and rapamycin treatment. We generated a high quality dataset byapplying a high resolution sampling, parallel sample processing and a dedicated data analysis pipeline that allowed us to select significant transient dynamics. By exploiting the temporal resolution of our data and the complementary of the shift experiments, we identified early phosphorylation changes regulated directly by TORC1 or by its proximal substrate kinases, and found both established and novel candidate TORC1 targets. Among the candidateTORC1 targets were the metabolic enzymes Amd1, Hom3, Nth1 and Tsl1, involved in nucleotide, amino acid and storage carbohydrate metabolism. Functional assessment of the role of phosphorylation for these and other enzymes was achieved by correlating phosphopeptide with the corresponding enzyme-associated metabolite dynamics. Candidate kinases regulating these enzymes were identified by establishing TORC1-proximity and differential kinase activity criteria. Our work provides first evidence of TORC1-dependent regulation of the metabolic enzymes Amd1and Hom3 by the kinases Sch9 and Atg1, respectively, providing mechanistic insights into on how TORC1 controls nucleotide and amino acid metabolism in response to the quality of the nitrogen source.
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Project description:To resolve the temporal sequence of phosphorylation events responding to nutritional and chemically induced up and downshifts of TORC1 signaling in S. cerevisiae wildtype, we performed quantitative MS phosphoproteomics upon nitrogen-quality up and downshifts and rapamycin treatment. We generated a high quality dataset byapplying a high resolution sampling, parallel sample processing and a dedicated data analysis pipeline that allowed us to select significant transient dynamics. By exploiting the temporal resolution of our data and the complementary of the shift experiments, we identified early phosphorylation changes regulated directly by TORC1 or by its proximal substrate kinases, and found both established and novel candidate TORC1 targets. Among the candidateTORC1 targets were the metabolic enzymes Amd1, Hom3, Nth1 and Tsl1, involved in nucleotide, amino acid and storage carbohydrate metabolism. Functional assessment of the role of phosphorylation for these and other enzymes was achieved by correlating phosphopeptide with the corresponding enzyme-associated metabolite dynamics. Candidate kinases regulating these enzymes were identified by establishing TORC1-proximity and differential kinase activity criteria. Our work provides first evidence of TORC1-dependent regulation of the metabolic enzymes Amd1and Hom3 by the kinases Sch9 and Atg1, respectively, providing mechanistic insights into on how TORC1 controls nucleotide and amino acid metabolism in response to the quality of the nitrogen source.
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