ABSTRACT: 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.
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.
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 (ΔrpaA). 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 ΔrpaA strain, we compared the time-averaged gene expression in the wild-type to the time-averaged gene expression in the ΔrpaA 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 ΔrpaA strain was constructed by pooling equal mass quantities of RNA from each ΔrpaA 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 ΔrpaA 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(ΔrpaA/wild-type) = 0.5 * (log2(ΔrpaA Cy3 / wild-type Cy5) - log2(wild-type Cy3 / ΔrpaA Cy5)), with the minus sign correcting for the sign changed caused by the dye swap). See supplementary file linked at foot of Series record.
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 ΔrpaA ΔkaiBC 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 ΔkaiBC 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 µM. 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.
Project description:Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation (ChIP) sequencing. We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts. We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription. RNA Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 RNA polymerase ChIP Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 Tiling Microarray of the cyanobacterium Synechococcus elongatus PCC 7942
Project description:The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-hour periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive – neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here we show that the topological status of the chromosome is highly correlated with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling responsive genes in E. coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle. Synechococcus elongatus PCC 7942 was subjected to two consecutive light/dark cycles and released into continuous light (T = 0). Cells were sampled every 4 hours from T = 24 to T = 84 hours for microarray analysis to characterize circadian gene expression. In a separate experiment, to characterize the response of S. elongatus to immediate chromosome relaxation, cells were sampled at T = 56 and T = 64 hours, immediately followed by novobiocin treatment (0.1 ug/ml), and the resulting response was measured by microarray after 5, 10, 30, 90, and 150 minutes. This experiment was designed to test whether chromosomal relaxation is sufficient to induce gene expression changes similar to those observed during the circadian cycle.
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 (ΔrpaA, ΔkaiBC) 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.
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.
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 ΔrpaA culture. Chromatin immunoprecipitation was performed on each sample from wht wild-type and from a pool of all samples from the ΔrpaA 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.
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.