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:The model cyanobacterium Synechocystis sp. PCC encodes nine different σ factors. SigA is the essential primary σ factor that is mainly responsible for expression of housekeeping genes during normal growth. Group 2 σ factors are non-essential in standard conditions, and actually all single, double, triple and quadruple inactivation strains of group 2 factors grow well in standard growth conditions. The roles of group 2 σ factors in oxidative stress acclimation were studied using the ΔsigBCDE strain of Synechocystis sp. PCC 6803 which is devoid of all group 2 σ factors, and triple inactivation strains, each containing one group 2 σ factor. The ΔsigBCD strain contains only SigE, ΔsigBCE only SigD, ΔsigBDE only SigC and ΔsigCDE only SigB. The expression of genes encoding known players of oxidative stress tolerance in the triple and quadruple mutants were compared in standard growth conditions using DNA microarray analysis.
Project description:The aim of this study was to find the cause for the previously reported inconsistency between oscillating transcription and constant protein levels under day-night growth conditions in cyanobacteria. To determine whether translational regulation counteracts transcriptional changes, Synechocystis sp. PCC 6803 was cultivated in an artificial day-night setting and the level of transcription, translation and protein was measured across the genome at different time points using mRNA sequencing, ribosome profiling and quantitative proteomics. This proteomics data set represents one layer of a larger data set covering three 'omics' levels.
Project description:Deciphering structure, function and mechanism of a new lysine methyltransferase cKMT1 in model cyanobacterium Synechocystis sp. PCC 6803
Project description:Quantification of circadian gene expression in WT Synechocystis sp. PCC 6803 cells We quantified circadian gene expression of the wild type Synechocystis PCC6083 strain. Over a 24 h time course, 6 samples for RNA isolation were taken at the following time points: 30 minutes before and after light is switched off (sample 1 - CT 11.5 and sample 2 - CT 12.5), 30 minutes before midnight (sample 3 - CT 17.5), 348 30 minutes before and after light onset (sample 4 - CT 23.5 and sample 5 - CT 0.5) and 30 minutes before noon (sample 6 - CT 5.5).
Project description:In eubacteria, replacement of one σ factor in the RNA polymerase (RNAP) holoenzyme by another one changes the transcription pattern. Cyanobacteria are eubacteria characterized by oxygenic photosynthesis and they typically encode numerous group 2 σ factors that closely resemble the essential primary σ factor. A mutant strain of the model cyanobacterium Synechocystis sp. PCC 6803 without functional group 2 σ factors (named as ΔsigBCDE) could not acclimate to heat, high salt, or bright light stress but in standard conditions ΔsigBCDE grew only 9% slower than the control strain. One-fifth of the genes in ΔsigBCDE were differently expressed compared to the control strain in standard growth conditions and several physiological changes in photosynthesis, and pigment and lipid compositions were detected. To directly analyze the σ factor content of RNAP holoenzyme in vivo, a His-tag was added to the γ subunit of RNAP in Synechocystis and RNAPs were collected. The results revealed that all group 2 σ factors were recruited by RNAP in standard conditions, but recruitment of SigB and SigC increased in heat stress, SigD in bright light, SigE in darkness and SigB, SigC and SigE in high salt, explaining the poor acclimation of ΔsigBCDE to these stress conditions. Cells from cyanobacteria Synechocystis sp. PCC 6803 named as control strain (CS) and a mutant strain without any functional group 2 sigma factors, ΔsigBCDE, were harvested (A730=1, 40 mL) directly from standard growth conditions (continuous illumination at the PPFD of 40 µmol m-2s-1, 32°C, ambient CO2). From three to four independent experiments were performed at each conditions.
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:Extracellular proteins are involved in a remarkable number of fundamental processes in cyanobacteria. Yet, there is limited knowledge regarding the identity and function of these proteins. Here, we introduce a solid-phase enhanced protein aggregation workflow that enables description of the cyanobacterial exoproteome with unprecedented depth. Application to cyanobacteria from three distinct habitats, Synechocystis sp. PCC 6803, Synechcoccus sp. PCC 11901 and Nostoc punctiforme PCC 73102, allowed the identification of up to 62% of all predicted secreted proteins. The approach was then extended to compare the Synechocystis sp. PCC 6803 wild-type secretome with that of a bloom-like aggregated state and a secretion-impaired mutant. Finally, we demonstrate that the workflow can be miniaturized and adapted to a 96-well format for high-throughput secretome analysis. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across species from different environments. Our approach can be applied to microbes from a wide range of habitats, with the potential to open new avenues of investigation in microbial exoproteomics.
Project description:The NDH1 complex fulfils numerous tasks in the cyanobacterial cell such as respiration, cyclic electron flow, and inorganic carbon concentration. Despite the immense progress in our understanding of structure/function relation of the cyanobacterial NDH1 complex, the subunits catalysing the NAD(P)H docking and oxidation are still missing. The gene sml0013 of Synechocystis 6803 encodes for a small protein of unknown function for that homologs exist in all completely known cyanobacterial genomes. The protein exhibits weak similarities to the NDF6 protein, which was reported from Arabidopsis chloroplasts as a NDH subunit (Ishikawa et al. 2008). A sml0013 inactivation mutant of Synechocystis 6803 was generated and characterized. It showed only weak differences regarding growth and pigmentation at various culture conditions; most remarkably it exhibited a glucose-sensitive phenotype in the light. The genome-wide expression pattern of the M-NM-^Tsml0013::Km mutant was almost identical to wild type when grown under high CO2 conditions as well as after shifts to low CO2 conditions. However, measurements of the photosystem I redox kinetic in cells of the M-NM-^Tsml0013::Km mutant revealed differences to wild type such as a decreased capability of cyclic electron flow as well as of utilization of electrons from catabolic processes. These results suggest that the Sml0013 protein (named NdhP) represent a novel subunit of the cyanobacterial NDH1 complex mediating its coupling to the respiratory or photosynthetic electron flow. Gene expression of Synechocystis sp. PCC 6803 WT and a M-NM-^Tsml0013::Km mutant was monitored at HC conditions (5% CO2) and at 24h after a shift to LC conditions (ambient air containing 0.035% CO2). Each condition was sampled in biological duplicates.