Project description:Application of genome-scale 'omics approaches to dissect subcellular pathways and regulatory networks governing the fast-growing response of Synechococcus sp. PCC 7002 response to variable irradience levels. We employed controlled cultivation and next-generation sequencing technology to identify transcriptional responses of euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 grown under steady state conditions at six irradiance levels ranging from 33 to 760 µmol photons m-2 sec-1.
Project description:This transcription profiling analysis of Chlorogloeopsis sp. PCC 9212 is designed to study the role of RfpA, RfpB, and RfpC in response to light color change Overall design: Chlorogloeopsis sp. PCC 9212 wild type and mutants cells were cultured in white light and far-red light with or without the supplement of erythromycin
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:Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803). Cultures were grown in medium modified with 5mM acrylic acid at pH 8 and compared to cultures grown in unmodified medium. Samples were processed in duplicate.
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: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 whole regulon of the LTTR All3953 was determined at 3 h after Ci deficiency in the cyanobacterium Anabaena sp. PCC 7120 by ChIP-Seq analysis. A TAP-tagged version of the protein was used for the chromatin immunoprecipitation. A total of 142 peaks were found, mainly located in the chromosome of Anabaena. Overall design: Two Anabaena sp PCC 7120 strains were constructed: one containing a TAP-tagged version of protein All3953 (strain CSS57), and a control strain expressing the TAP-tag under the all3953 promoter (strain CSS68). Cells of the two strains were grown in BG110 medium supplmented with 6 mM NH4Cl and 10 mM NaHCO3, and bubbled with 1% CO2-containing air. When the cultures had reached exponential phase (3-5 µgChl/ml), the cells were collected, washed with BG110, and resuspended in the same volume of BG110 supplemented with 6 mM NH4Cl and bubbled with air (low Ci conditions) for 3 h. Chromatin immunoprecipitation was carried out using extracts of both strains and IgG-conjugated dynabeads, and the resultant DNA was subjected to massive sequencing. The Triform algorithm method (Kornacker et al, 2013) was used to analyze the data. To identify the specific peaks found in strain CSS57, the CSS68 (control strain) ChIP data was used as the background for analysis of the CSS57 ChIP data.
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)