Differential expression of whole transcriptome in Synechocystis sp. PCC 6803
ABSTRACT: The model cyanobacterium Synechocystis sp. PCC 6803 was used for a systematic survey of differential expression with a focus on antisense (as)RNAs and non-coding (nc)RNAs. A microarray was constucted with on average 5 probes for each transcript known thus far, including ncRNAs and asRNAs. The resulting 20,431 individual probes are duplicated on the array (Agilent 4x44k custom array) representing a technical replicate. Hybridization of this array with total RNA isolated from cultures raised under different growth conditions identified transcripts from intergenic spacers and in antisense orientation to known genes (natural cis-asRNAs) with differential expression compared to control hybridizations. This shows the involvement of such transcripts in the regulation of adaptation to various stresses. 12 RNA hybridizations (1 control & 3 stress conditions, 3 times each)
Project description:The model cyanobacterium Synechocystis sp. PCC 6803 was used for a systematic survey on the number and types of antisense (as)RNAs. A tiled microarray was constructed with probes for both strands of the genes or intergenic spacers with candidate transcripts predicted by an algorithm and an equally sized and distributed control set. The resulting 102,739 individual probes cover an accumulated length of 1,441,146 nt or about 40% of the total chromosome sequence of Synechocystis 6803. Hybridization of this array with total RNA isolated from cultures raised under different growth conditions identified a high number of transcripts from intergenic spacers and in antisense orientation to known genes (natural cis-asRNAs). Extrapolated to the whole genome, around 10% of all open reading frames in Synechocystis 6803 may have a corresponding asRNA, suggesting a much more important role of chromosomally encoded asRNAs in bacteria then anticipated so far. Keywords: stress response 4 RNA hybridizations, 2 DNA controls. The raw data from the DNA hybridizations are in the GSE14410_DNA_1.txt and GSE14410_DNA_2.txt files.
Project description:Comparison of gene expression of WT Synechocystis cells with cells overexpressing the sRNA PsrR1 in order to detect direct targets of the sRNA PsrR1. We monitored gene expression of an Synechocystis PCC6083 overexpressor strain (psrR1+) (Mitschke et al., 2011) harboring a self-replicating plasmid from which PsrR1 is transcribed under control of the copper responsive PpetJ promoter and a control strain (WT) harboring an empty plasmid 0h and 24 h after copper depletion. For the timepoint (0h) we sampled biological replicates and for timepoint (24h) we sampled biological triplicates.
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 (ΔssaA) 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 ΔssaA 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 ∆ssaA 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.
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 ‘least oscillating set’ 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.
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:Iron is an essential cofactor in many metabolic reactions. Mechanisms controlling iron homeostasis need to respond to changes in extracellular conditions, but must also keep the concentration of intracellular iron under strict control, as free ferrous iron (Fe2+) can lead to the generation of reactive oxygen species. Due to its role as redox carrier in photosynthesis, the iron quota in cyanobacteria is about 10 times higher than in model enterobacteria, but the molecular details how such high quota is tightly regulated have remained obscure. Here, we measured time-resolved gene expression changes after iron depletion in the cyanobacterium Synechocystis sp. PCC 6803 using a comprehensive microarray platform monitoring both protein-coding and non-coding transcripts. In total, 644 protein-coding genes were differentially expressed during the first 72h. Many of these proteins are associated with iron transport, photosynthesis or ATP synthesis. Comparing our data with three previous studies, we identified a core set of 28 genes involved in the iron stress response. Among them were genes encoding proteins important for assimilation of inorganic carbon, suggesting a previously unknown link between the carbon and iron regulatory networks. Nine of the 28 genes are of unknown function and constitute key targets for detailed functional analysis. Applying identical statistical and clustering criteria as for the protein-coding fraction, we also identified 10 small RNAs, 62 anti-sense RNAs, four 5’UTRs and 7 intergenic elements as likely to be involved in the iron regulatory network. Hence, our genome-wide profiling results indicate an unprecedented complexity in the iron-related regulatory network of cyanobacteria. We monitored iron limitation stress induced gene expression changes in the cyanobacterial model organism Synechocystis sp. PCC 6803. We included a control sample without iron-stress (0h) and 5 samples from timepoints after stress induction (3h, 12h, 24h, 48h, 72h). Each timepoint was sampled from two independent biological replicates.
Project description:Every cyanobacterial species contains gene encoding site-2-protease (S2P) homolog. The studied prokaryotic S2P homologs play essential roles in regulating stress response through intramembrane proteolysis of membrane-bound anti-sigma factors. Here, gene of Slr0643, one of four S2P homologs in Synechocystis sp. PCC 6803, was insertionally disrupted to explore its physiological role. Only a partially segregated mutant was obtained, indicating its indispensability for growth. The partially disrupted mutant could not grow at pH 6.5, while wild type acclimated to pH 6.5 quickly. The slr0643 gene expression was transiently induced after pH transfer from 7.5 to 6.5. Both evidences demonstrated the pivotal role of fully functional Slr0643 in acid acclimation. DNA microarray and quantitative RT-PCR analyses decoded genes involved in early acid acclimation and revealed differentially expressed genes due to slr0643 disruption at both pH conditions. Early acid acclimation to pH 6.5 included upregulation of sigH, hik16 and hik35, and downregulation of pcrR and sigG; as well as downregulation of porins and upregulation of inorganic carbon and nitrogen transporters. Defective photosynthesis and excess expression of NADH dehydrogenase, together with over upregulation of carbon transporter and repression of nitrogen transporter and metabolism gene contributed to the acid lethality of mutant at pH 6.5. Most interestingly, analysis of microarray data revealed the close relationship between slr0643 disruption and expression of sigH operon. Therefore it was implied that Slr0643/Sll0857/SigH might acts through S2P/anti-Sigma factor/Sigma factor mechanism to play a role in acid acclimation. Loop design was used to compare differential expression of wild type and mutant at pH 7.5 and pH 6.5 respectively, including dye-swape. Biological variation was sampled by extracting RNA from three independent experiments and pooling them together before hybridizations. Two or four replicate chips for each comparison and two replicate printings per chip represent technical repeats.
Project description:RNA samples from cells collected at twenty four time points in 48-hours diurnal cycles were analyzed, and they compared between each other for transcriptional profiles of global genes 24 RNA samples at different time points will be compared for this cycling condition. An equimolar mixture of RNA from all time points was used as the control.
Project description:Small CAB-like proteins (SCPs) are single-helix light-harvesting-like proteins found in all organisms performing oxygenic photosynthesis. We investigated the function of these stress-induced proteins in the cyanobacterium Synechocystis sp. PCC 6803 by comparing a strain, which expresses SCPs constitutively, with a mutant lacking all five SCPs. In the absence of SCPs, cells were larger, and showed irregular thylakoid structure and cell-surfaces. Deletion of scp genes strongly affected the carbon-nitrogen balance, causing accumulation of carbohydrates and a decrease in N-rich compounds (proteins and chlorophyll a). Data from transcriptomic and metabolomic experiments demonstrated that SCPs mediated stabilization of chlorophyll a under stress conditions is crucial to maintain the C/N balance. SCPs diminished the formation of reactive oxygen species, preventing cellular damage by adjusting the de-novo production of chlorophyll a to demand levels. Lack of SCP expression under stress conditions had a large impact on the metabolism of the entire cell. Synechocystis 6803 PSI-less (Shen et al 1993) and PSI-less/ScpABCDE- (Xu et al 2004) mutants were cultivated at 30°C at a light intensity of 4–5 μmol photons m–2 s–1 in BG-11 growth medium (Rippka et al 1979) supplemented with 10 mM glucose. Cells (40–50 ml) from both control and the mutant cultures were collected at mid-log phase by rapid filtration (Pall Supor 800 Filter, 0.8 mm) and total RNA extracted after Pinto et al. (Pinto et al 2009). Single-stranded cDNA preparation, labelling, and hybridization onto the microarray slides were performed after Eisenhut et al. (2007). The microarray slides were scanned with an Agilent Microarray Scanner (model G2505B) with default settings.
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.