Project description:Like many other organisms, cyanobacteria exhibit rhythmic gene expression with a period length of 24 hours to adapt to daily environmental changes. In the model organism Synechococcus elongatus PCC 7942 the central oscillator consists of three proteins: KaiA, KaiB and KaiC and utilizes the histidine kinase SasA and its response regulator RpaA as output-signaling pathway. Synechocystis sp. PCC 6803 contains two additional homologs of the kaiB and kaiC genes. Here we demonstrate that RpaA interacts with the core oscillator KaiAB1C1 of Synechocystis sp. PCC 6803 via SasA, similar to Synechococcus elongatus PCC 7942. However, interaction with the additional Kai homologs was not detected, suggesting different signal transduction components for the clock homologs. Inactivation of rpaA in Synechocystis sp. PCC 6803, lead to reduced viability of the mutant in light-dark cycles that aggravated under mixotrophic growth conditions. Chemoheterotrophic growth in the dark was abolished completely. In accordance, transcriptomic data revealed that RpaA is involved in the regulation of genes related to CO2‑acclimation and carbon metabolism under diurnal light conditions. Further, our results indicate that RpaA functions in the posttranslational regulation of glycogen metabolism as well, and a potential link between the circadian clock and motility was identified.
Project description:In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits gyrA, gyrB and overexpression of topoisomerase I (TopoI) topA and analyzed the transcriptional response to gyrase knock-downs (endpoint in triplicate) and topoisomerase I overexpression (endpoint in triplicate, and 19 time points time series before and after induction) in Synechocystis sp. PCC 6803 via RNA-seq of coding RNA. In detail, Illumina Ribo-Zero Plus rRNA Depletion Kit was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was evaluated with the RNA Pico 6000 kit on the Agilent 2100 Bioanalyzer. RNA was free of detectable rRNA. Preparation of cDNA libraries was performed according to the manufacturer’s instructions for the TruSeq stranded mRNA kit (Illumina, San Diego, CA, United States). Subsequently, each cDNA library was sequenced on an Illumina NextSeq 500 system (2 x 75 nt PE high output v2.5).
Project description:In contrast to Synechococcus elongatus PCC 7942, which has been the model cyanobacterium for the study of the prokaryotic circadian clock for more than 20 years, only few data exist on the circadian behaviour of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiABC operon present in this organism was shown to encode a functional KaiC protein which interacts with KaiA, similar to the Synechococcus elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect in light-dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown in constant light. Microarray experiments performed with cells grown for one day in a light-dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison to the wild type. Most interestingly, we identified genes like the gene encoding the cyanobacterial phytochrome Cph1 and the light repressed protein LrtA as well as several hypothetical open reading frames with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison to the wild type. In addition, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark.
Project description:In contrast to Synechococcus elongatus PCC 7942, which has been the model cyanobacterium for the study of the prokaryotic circadian clock for more than 20 years, only few data exist on the circadian behaviour of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiABC operon present in this organism was shown to encode a functional KaiC protein which interacts with KaiA, similar to the Synechococcus elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect in light-dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown in constant light. Microarray experiments performed with cells grown for one day in a light-dark cycle revealed many differentially regulated genes with known functions in the M-NM-^TkaiABC mutant in comparison to the wild type. Most interestingly, we identified genes like the gene encoding the cyanobacterial phytochrome Cph1 and the light repressed protein LrtA as well as several hypothetical open reading frames with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in M-NM-^TkaiABC cells in comparison to the wild type. In addition, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Three timepoints with two samples (WT and Mutant). Two replicates for each timepoint/sample. RNA hybridization.
Project description:To investigate acclimation mechanisms employed under extreme high light conditions, gene expression analysis was performed using the model microalgae Synechocystis sp. PCC 6803 (PCC 6803) cultured under various light intensities. From the low to the mid light conditions, the expression of genes related to light harvesting systems was repressed, whereas that of CO2 fixation and of D1 protein turnover-related genes was induced. Gene expression data also revealed that the down-regulation of genes related to flagellum synthesis (pilA2), pyridine nucleotide transhydrogenase (pntA and pntB), and sigma factor (sigA and sigF) represents acclimation mechanisms of PCC 6803 under excessive high light conditions.
Project description:We investigated Synechocystis high-density microarray data of three high-resolution time-series experiments with alternating light/dark rhythm, transition to continuous light, and transition to continuous darkness. Using M-bM-^@M-^Xleast oscillating setM-bM-^@M-^Y normalization and a clustering approach we found a daily temporal program for rhythmic expression of protein-coding and non-coding genes under light/dark conditions. All rhythms, however, damped out rapidly under continuous conditions. We quantified gene expression of wild type Synechocystis PCC 6083 over 48 hours under three different light regimes; 12:12-h light/dark cycle (LDLDL), transfer to continuous light (LDLLL), and transfer to continuous dark (LDDDD). Samples were taken every two hours starting at CT 5.5 with an extra sample 30 minutes after dawn. Before the transfer to continuous light/dark in the LDLLL and LDDDD experiment, samples were taken every 6 with an extra sample 30 minutes after dawn.