Project description:Ethylene is a gaseous signal sensed by plants and bacteria. Heterologous expression of the ethylene-forming enzyme (EFE) from Pseudomonas syringae in cyanobacteria leads to the production of ethylene under photoautotrophic conditions. The recent characterization of an ethylene responsive signaling pathway affecting phototaxis in the cyanobacterium Synechocystis sp. PCC 6803 implies that biotechnologically relevant ethylene synthesis may induce regulatory processes which are not related to changes in the metabolism. Here we provide data that endogenously produced ethylene accelerates movement of cells towards light. Microarray analysis demonstrates that ethylene deactivates transcription from the csiR1/lsiR promoter which is under control of the two-component system consisting of the ethylene and UV-A-sensing histidine kinase UirS and the DNA-binding response regulator UirR. Surprisingly, only very few other transcriptional changes were detected in the microarray analysis providing no direct hints to possible bottlenecks in phototrophic ethylene production. Overall design: We performed whole-genome transcript profiling of an ethylene producing strain of Synechocystis sp. PCC 6803.
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. Overall design: The gene expression in Synechocystis sp. PCC 6803 was analyzed following culture at 30, 50, 300, 1000, 1100, and 1300 μmol m-2 s-1. Three independent experiments were performed under each light intensity. To check the repeatability, some RNA samples are analyzed in three microarraies.
Project description:Acclimation to low CO2 conditions in cyanobacteria involves the coordinated regulation of genes mainly encoding components of the carbon concentration mechanism (CCM). Making use of several independent microarray datasets a core set of CO2-regulated genes was defined for the model strain Synechocystis sp. PCC 6803. On the transcriptional level, the CCM is mainly regulated by the well-characterized transcriptional regulators NdhR and CmpR, whereas the role of an additional regulatory protein, namely cyAbrB2 belonging to the widely distributed AbrB regulator family that was originally characterized in the genus Bacillus, is less defined. Here we present results of transcript profiling of the wild type and a ΔcyabrB2 mutant of Synechocystis sp. PCC 6803 after shifts from high CO2 (5% in air, HC) to low CO2 (0.04%, LC). Evaluation of the transcriptomic data revealed that cyAbrB2 is involved in the regulation of several CCM-related genes such as sbtA/B, ndhF3/ndhD3/cupA and cmpABCD under LC conditions, but apparently acts supplementary to the main regulators. Under HC conditions, cyAbrB2 deletion changes the expression of photosystem II subunits, light-harvesting components, and Calvin-Benson-Bassham cycle enzymes. Overall design: We analyzed gene expression in Synechocystis sp. PCC 6803 WT as well as a ΔcyabrB2 mutant at HC (5% CO2) and 24h after shift to LC (air). For the WT six biological replicates were analyzed for each sampling point; the ∆cyAbrb2 mutant was analyzed in duplicates.
Project description: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 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. Overall design: 12 RNA hybridizations (1 control & 3 stress conditions, 3 times each)
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. Three timepoints with two samples (WT and Mutant). Two replicates for each timepoint/sample. RNA hybridization.
Project description:The responses of the transcriptome were monitored in Synechocysis PCC 6803 during a linear rate of evaporation of the culture to dryness (desiccation). For each time point, total RNA were isolated from stressed and unstressed cells, reverse-transcribed, differentially labelled (dye swapped), hybridized together (stressed versus unstressed samples) and analyzed with DNA glass microarrays (two slides per each time point) (Custom-commercial array : CyanoCHIP version 2.0, TAKARA). To identify differentially expressed genes, the median of the normalized ratio of Cy5/Cy3 intensity was calculated for each spot of the replicated dye-swap. The results of the analysis were carefully examined to exclude the dye effect between the 2 Cy-swapped arrays. Keywords: Dehydration, stress response, time course, transcription, cyanobacteria Overall design: Goal - Compare the responses of cyanobacterium Synechocystis sp. PCC 6803 to UV-irradiation and dehydration/desiccation stress. The data here relate to dehydration/desiccation stress. Origin of biological sample - The axenic strain Synechocystis sp. PCC 6803 was obtained from the American Type Culture Collection (ATCC 27184). Special attention was given to maintain all the Synechocystis cultures under the same conditions to minimize environmental variation. Brief description - Ultraviolet (UV) light and desiccation stresses often co-occur in natural environments, making their combined effects important selective factors within microbial populations. The effects of these stresses on the cyanobacterium Synechocystis sp. PCC 6803 was assessed through transcriptional analyses using differential display and microarray assays. Experimental factors and design - Axenic Synechocystis cultures were grown at 25°C in BG-11 medium, on a rotary shaker at 70 rpm, at a photon flux density of 200 µmol photons m-2 s-1. At the midpoint of the exponential phase of growth (~ 10^6 to 10^7 cells ml-1), 25-ml aliquots of axenic cultures were transferred to glass Petri dishes with covers and pre-adapted overnight. The covers were removed and cultures were subjected to a linear rate of evaporation of (55% relative humidity maintained in the incubator). Samples were taken at 25, 20, 15, 12, 7, 3 and 0.5 ml of evaporated cultures and completely dried cultures; the latter corresponding to a total of 15 h of dehydration. As controls, RNA was obtained from Synechocystis cells grown without dehydration. Cells were disrupted with glass beads and RNA was extracted with a buffer of acidified phenol (65°C), sodium acetate pH 5.1, EDTA and sodium dodecyl sulfate), followed by ethanol precipitation. The RNA (10 µg) was annealed with 20 µg Random RT primer and reverse transcription into cDNA was achieved with Superscript II reverse transcriptase enzyme for 2h, at 42°C. The assay contained dGTP, dCTP, dATP and dTTP, each a final concentration of 0.5 mM. After hydrolysis of RNA with 0.1M NaOH and 10 mM EDTA, then neutralization, cDNAs were purified using a Quiagen QIAquick PCR purification kit as modified in the Genisphere 3DNA Array 350RP protocol. Two experimental replicates of RNAs from time points 12 ml, and two experimental replicates from 0.5 ml, of dehydration, were used for hybridization to microarrays.
Project description:Functional analysis of engineered carbon limitation in the ∆ndhD3/ndhD4/cmpA/sbtA (= ∆4) Synechocystis mutant reveals a global phenocopy of wild type acclimation to low CO2 and multi-layered Ci-regulation. Overall design: We analyzed gene expression in Synechocystis sp. PCC 6803 WT as well as a ∆ndhD3/ndhD4/cmpA/sbtA mutant (= ∆4) at HC (5% CO2) as well as 3h and 24h after shift to LC (air). For each strain and each sampling point, 2 biological replicates were analyzed.