Circadian control of global gene expression by the cyanobacterial master regulator RpaA
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ABSTRACT:
ORGANISM(S): Synechococcus elongatus PCC 7942
SUBMITTER: Joseph Markson
PROVIDER: E-GEOD-50922 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The cyanobacterium Synechococcus elongatus contains a circadian clock which coordinates circadian changes in gene expression of a large percentage of its genes. The response regulator RpaA has been implicated as an important regulator of many circadian genes, but the role of this protein in regulating changes in gene expression genome-wide is not known. We show that deletion of rpaA abrogates circadian gene expression genome-wide and arrests cells in a gene expression state highly similar to that of wildtype cells in the morning. Furthermore, we show that RpaA binds DNA in an circadian manner that is dependent on phosphorylation of the protein. To demonstrate the sufficiency of phosphorylated RpaA in driving global changes in gene expression, we used RNA sequencing to measure changes in gene expression elicited by a phosphomimetic of RpaA (RpaA D53E) and compared these changes to those that occur during a circadian cycle in wildtype cells. This analysis reveals that induction of RpaA D53E is sufficient to drive all circadian gene expression changes that happen from dawn to dusk in wildtype cells. Interestingly, the dynamics of gene expression elicited by RpaA D53E induction mirror those observed during a circadian cycle in wildtype cells, suggesting that the dynamics of circadian gene expression and hard-wired in the regulon downstream of RpaA. Enriched mRNA was prepared from synchronized wildtype S. elongatus cells every four hours over a circadian cycle and sequenced using the Illumina TruSeq Stranded mRNA Sample Prep Kit and Illumina HiSeq technology. To test the role of phosphorylated RpaA in driving circadian gene expression, we generated a strain which we refer to as OX-D53E that lacks core clock components (M-NM-^TrpaA, M-NM-^TkaiBC) with an RpaA phosphomimetic (RpaA D53E) under the control of an IPTG-inducible promoter (Ptrc::rpaAD53E). We used the same methodology to measure gene expression in OX-D53E before and after induction of RpaA D53E. As a control, we also measured gene expression in the OX-D53E strain over time in the absence of IPTG. Also, we generated a strain similar to OX-D53E in which the only difference was that no gene was inserted downstream of the IPTG inducible promoter (OX-Empty). We measured gene expressio in OX-Empty before and after IPTG addition to test for off-target effects of IPTG.
2013-12-04 | E-GEOD-51112 | biostudies-arrayexpress
Project description:The goal of the experiment was to determine whether gene expression oscillates in the absence of rpaA. It was reported previously (e.g., Takai et al, PNAS 2006) that activity of a handful of individual expression reporters was arrythmic, and we conducted this microarray timecourse to determine whether expression is arrhythmic geneome-wide. Cultures were grown in a turbidostat as described previously (Vijayan et al, PNAS 2009). Cultures were entrained with two consecutive light/dark cycles and released into continuous light at time T = 0. Cultures were samples every 4 hours for 48 h between T = 24 h and T = 72 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray.
2013-12-04 | E-GEOD-50908 | biostudies-arrayexpress
Project description:The goal of the experiment was to determine the difference in gene expression between the wild-type strain and a strain lacking rpaA (M-NM-^TrpaA). Because gene expression is not at steady-state in the wild-type -- it oscillates with a circadian period -- and we did not know a priori whether it is at steady-state in the M-NM-^TrpaA strain, we compared the time-averaged gene expression in the wild-type to the time-averaged gene expression in the M-NM-^TrpaA strain. Cultures were grown in a turbidostat as described previously (Vijayan et al, PNAS 2009). Cultures were entrained with two consecutive light/dark cycles and released into continuous light at time T = 0. Cultures were samples every 4 hours for 20 h between T = 24 h and T = 44 h, inclusive (no 4 h sample was acquired because, in a circadianly-oscillating culture, it would be duplicative with the 24 h timepoint). A pool of RNA representing time-averaged wild-type RNA was constructed by pooling equal mass quantities of RNA from each wild-type timepoint. A pool of RNA representing time-averaged RNA for the M-NM-^TrpaA strain was constructed by pooling equal mass quantities of RNA from each M-NM-^TrpaA timepoint. These two pooled RNA samples were compared by two-color Agilent microarray. To correct for dye biases, two microarrays were performed -- one in which the M-NM-^TrpaA pool was labeled with Cy3 and the wild-type pool was labeled with Cy5, and another in which the dyes were swapped. In the manuscript, the average log2 ratio value from these two microarrays was employed (average of log2(M-NM-^TrpaA/wild-type) = 0.5 * (log2(M-NM-^TrpaA Cy3 / wild-type Cy5) - log2(wild-type Cy3 / M-NM-^TrpaA Cy5)), with the minus sign correcting for the sign changed caused by the dye swap). See supplementary file linked at foot of Series record.
2013-12-04 | E-GEOD-50919 | biostudies-arrayexpress
Project description:The goal of the experiment was to obtain a replicate of the wild-type LL circadian timecourse published in Vijayan et al, PNAS 106: 22564-22568 (2009), in order to identify reproducible circadian genes in LL. Cultures were grown in a turbidostat as described previously (Vijayan et al, PNAS 2009), except that the culture volume was 3 L instead of 4.5 L. Cultures were entrained with two consecutive light/dark cycles and released into continuous light at time T = 0. Cultures were samples every 4 hours from T = 36 h and T = 64 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray. Timepoint T = 52 h is omitted due to poor data quality.
2014-03-06 | E-GEOD-52486 | biostudies-arrayexpress
Project description:The goal of this experiment was to determine whether global circadian gene expression oscillation depends strictly on the presence of rpaA, even when the KaiABC post-translational oscillator is oscillating with a circadian period. Strains deleted for rpaA lack functional KaiABC post-translational oscillators because their reduced kaiBC expression level leads to a non-permissive Kai protein stoichiometry. We restored KaiABC post-translational oscillator function in a M-NM-^TrpaA M-NM-^TkaiBC strain by ectopic expression of kaiBC from the Ptrc promoter and used microarrays to measure the timecourse of gene expression globally. As a control, we used microarrays to measure the gene expression timecourse in a M-NM-^TkaiBC Ptrc::kaiBC strain, in which gene expression was expected to be rhythmic (Y Murayama et al, J. Bac. 198, 2008), as it is in the pure wild-type strain. Cultures were grown in a flasks bubbled with 1% CO2 in air, initially in the absence of IPTG. Cultures were treated with two consecutive light/dark cycles and released into continuous light at time T = 0, at which time IPTG was added to a final concentration of 6 M-BM-5M. Cultures were samples every 4 hours for 44 h between T = 24 h and T = 68 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray.
2013-12-04 | E-GEOD-50920 | biostudies-arrayexpress
Project description:The response regulator RpaA is required for control of genome-wide gene expression by the cyanobacterial circadian clock. RpaA is predicted to be a DNA binding protein based on sequence homology, but prior studies have been unable to detect binding in vitro or in vivo to a small panel of promoters. We used ChIP-Seq to determine whether RpaA associates with DNA in vivo, and if so, with what dynamics. We find that RpaA binds to over 100 location in the genome in a circadian manner, with strongest binding occuring around subjective dusk. Analysis of these binding sites shows that RpaA directly regulates the expression of clock components to generate feedback on the core oscillator, and also regulates expression of a small set of circadian effectors that in turn orchestrate global expression rhythms. Crosslinked samples were acquired every four hours from a turbidostatic wild-type (AMC408) cultures in constant light (LL) following entrainment with two light-dark (LD) cycles. As a negative control, we acquired samples similarly from an M-NM-^TrpaA culture. Chromatin immunoprecipitation was performed on each sample from wht wild-type and from a pool of all samples from the M-NM-^TrpaA culture. Libraries were prepared from the ChIP samples and sequenced with Illumina technology. For the wild-type, biological replicate samples were acquired at times of maximum and minimum RpaA binding.
2013-12-04 | E-GEOD-51093 | biostudies-arrayexpress
Project description:We employed chromatin pull-down and deep sequencing to globally identify HetR DNA targets in vivo at 6 hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Chromatin pull down of hetR mutant strain UHM103 carrying pAM4375, which expresses HetR-6xHis, and a wild-type control. One dataset was collected.
2013-10-31 | E-GEOD-51886 | biostudies-arrayexpress
Project description:Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors and gels derived from alkoxide precursors and incubated under constant light for 24 hours prior to RNA extraction. Cultures suspended in liquid media were also exposed to 500 mM salt stress and incubated under identical conditions, for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change >/= 1.5 and P-value < 0.01), the expression of 1143 genes were significantly altered by encapsulation in alkoxide derived gels, and only 243 genes were common to both encapsulation chemistries. Additional qRT-PCR analyses of four select genes; ggpS, cpcG2, slr5055, and sll5057, confirmed microarray results. These results illustrate that encapsulation stress is quite different than salt stress in terms of gene expression response. Furthermore, a number of hypothetical and unknown proteins associated with encapsulation and alcohol stress have been identified, with implications for improving encapsulation protocols and rationally engineering microorganisms for direct biofuel production. 16 samples; 4 biological replicates each of 4 treatments
2012-04-23 | E-GEOD-37482 | biostudies-arrayexpress
Project description:Protein phosphorylation via serine/threonine protein kinases (Spk) is a widespread mechanism to adjust cellular processes toward changing environmental conditions. To study their role(s) in cyanobacteria, we established a collection of 11 completely segregated spk mutants among the 12 annotated Spk’s in the model cyanobacterium Synechocystis sp. PCC 6803. Screening of the mutant collection revealed that especially the mutant defective in SpkB encoded by slr1697 showed clear deviations compared to wild type (WT) regarding carbon metabolism, i.e., a reduced growth rate at low CO2 and in the presence of glucose, different glycogen accumulation patterns, and a higher tolerance to external H2O2 than the WT. The proteome of ∆spkB showed several distinct differences compared to WT, which indicate changes of the cell surface but also metabolic functions. A phospho-proteome analysis revealed decreased phosphorylation of the carboxysome-associated protein CcmM and the regulatory PII protein in the mutant compared to WT, whereas the allophycocyanin alpha subunit was stronger phosphorylated. The decreased phosphorylation of PII was verified in Western-blot experiments, indicating a clearly delayed PII phosphorylation in cells shifted from nitrate-containing to nitrate-free medium. Furthermore, the mutant ∆spkB showed differences in the state transition consistent with the changed phosphorylation of allophycocyanin. Collectively our results indicate that SpkB is an important regulator under different environmental conditions in Synechocystis and seems to interact in the PII phosphorylation and probably with further substrates in a kinase network.
2024-01-26 | PXD040383 | Pride
Project description:The general acclimation of cyanobacteria to low carbon (LC) conditions includes coordinated alterations of gene expression and metabolism. To analyze possible signals for LC sensing and compensating reactions, we compared wild-type (WT) cells with two mutants of Synechocystis, the carboxysome-less mutant ccmM and the photorespiratory mutant ΔglcD1/D2. Metabolic phenotyping revealed that the mutant ΔccmM accumulated high 2-phosphoglycolate (2PG) levels while the ΔglcD1/D2 mutant accumulated glycolate, indicating oxygenase activity of RubisCO at high carbon (HC). The changes in the metabolite spectrum were compared to alterations in the global gene expression pattern. Cells of HC-grown mutants ΔccmM and ΔglcD1/D2 showed altered mRNA levels for many genes involved in photosynthesis, high light stress, and N-assimilation, while LC-specific genes such as those for inorganic carbon (Ci) transporters were not increased. After a shift to LC, mutant ΔglcD1/D2 revealed gene expression changes similar to WT cells, while mutant ΔccmM showed no differential expression of most LC-induced genes under identical conditions. In fact, none of the genes for Ci transporters or other components of the carbon concentrating mechanism (CCM) displayed higher transcript levels in the ΔccmM mutant. This finding renders a direct role for 2PG as a metabolic signal component for the induction of CCM during LC acclimation less likely. Because, the transcription pattern of ΔglcD1/D2 under LC showed specific differences compared to WT, a potential role for glycolate as a signal molecule that may trigger expression of parts of the CCM is proposed. Transcriptional profiling of carboxysomal and photorespiratory mutants of Synechocystis sp. PCC 6803 under high carbon (HC) and low carbon (LC) conditions relative to the wildtype response.
2011-12-30 | E-GEOD-31672 | biostudies-arrayexpress