Project description:Cyanobacteria play pivotal roles in global biogeochemical cycles through oxygenic photosynthesis. To maintain cellular homeostasis, these organisms employ sophisticated acclimation mechanisms to adapt to environmental fluctuations, particularly nitrogen availability. While nitrogen deprivation triggers dormancy, excess ammonium exerts toxic effects on cyanobacteria and other photosynthetic organisms - a phenomenon whose acclimation mechanisms remain poorly understood. TurboID based proximity labeling coupled with quantitative proteomics revealed a robust set of putative Sll0528 interacting proteins.
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.
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: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.
Project description:The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model system for studying biochemistry, genetics and molecular biology of photobiological processes. Despite its importance in basic and applied research, the genome-wide picture of transcriptional regulation in this bacterium is limited. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. We designed, validated and used Synechocystis genome-wide oligonucleotide (70-mer) microarray (representing 96.7% of all chromosomal ORFs) to study transcriptional activity of the cyanobacterial genome in response to S deprivation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate withdrawal were relatively moderate, but significant and consistent with growth kinetics; 2) S acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems I and II, cytochrome b6/f, and ATP synthase indicated reduced light-harvesting and photosynthetic activity; 3) S deprivation elicited transcriptional responses associated with general growth arrest and stress; 4) A large number of genes regulated by S availability encode hypothetical proteins or proteins of unknown function; 5) Hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. The expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful sulfur to no sulfur, Synechocystis undergoes coordinated transcriptional changes, including genes whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient limitation was dominated by decrease in abundances of many transcripts. However, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. Down-regulation of transcripts encoding proteins whose function depends on a cellular sulfur status indicated that the observed repression has a specific regulatory component. The repression of certain sulfur-related genes was paralleled by activation of genes involved in internal and external S scavenging. Keywords: stress response, time course
Project description:Cyanobacteria, photoautotrophic prokaryotes, contribute significantly to the global photosynthesis and require large amounts of the essential micronutrient iron in order to maintain their Fe-rich photosynthetic apparatus. Here we use the model organism Synechocystis sp. PCC 6803 (later referred to Synechocystis) in both, standard and iron stress conditions, to study transcription and post-transcription regulation in iron deprivation. Although iron is one of the most abundant metals on earth, it is not soluble under aerobic conditions. Thus Synechocystis had to find ways to overcome iron deficiency. At the same time, however, free intracellular iron needs to be kept at permissive levels, as it becomes toxic under aerobic conditions by producing reactive oxygen species. For these reasons, complex regulatory networks have evolved to tightly control intracellular iron concentrations, ensuring its essential function yet avoiding cellular damage (Pierre Cornelis et al). In a previous study, we investigated iron deprivation in Synechocystis using customised amplification library for the analysis of global gene expression in the unicellular cyanobacterium (Hernández-Prieto et al, 2012; Georg et al, 2017),(Georg et al, 2017) but it is still little known about RNA stability in this organism. We now extend this study through a transcriptome wide half-life analysis in Synechocystis grown under standard and iron-limiting conditions using oligonucleotide microarrays that detect both protein-coding and non-coding transcripts (ncRNA). We used the antibiotic Rifampicin to stop the transcription. Samples were taken at time points 0 min (before the addition of rifampicin) and in a time series of 2 min, 4 min, 8 min, 16 min, 32 min and 64 min after its addition.
Project description:The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model system for studying biochemistry, genetics and molecular biology of photobiological processes. Despite its importance in basic and applied research, the genome-wide picture of transcriptional regulation in this bacterium is limited. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. We designed, validated and used Synechocystis genome-wide oligonucleotide (70-mer) microarray (representing 96.7% of all chromosomal ORFs) to study transcriptional activity of the cyanobacterial genome in response to S deprivation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate withdrawal were relatively moderate, but significant and consistent with growth kinetics; 2) S acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems I and II, cytochrome b6/f, and ATP synthase indicated reduced light-harvesting and photosynthetic activity; 3) S deprivation elicited transcriptional responses associated with general growth arrest and stress; 4) A large number of genes regulated by S availability encode hypothetical proteins or proteins of unknown function; 5) Hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. The expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful sulfur to no sulfur, Synechocystis undergoes coordinated transcriptional changes, including genes whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient limitation was dominated by decrease in abundances of many transcripts. However, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. Down-regulation of transcripts encoding proteins whose function depends on a cellular sulfur status indicated that the observed repression has a specific regulatory component. The repression of certain sulfur-related genes was paralleled by activation of genes involved in internal and external S scavenging. Keywords: stress response, time course Synechocystis sp. PCC 6803 was grown photoautotrophically in BG-11 medium supplemented with 8mM NaHCO3 and buffered with 10mM HEPES (pH 7.4). The cells were grown in 250ml flasks at 32oC under a light intensity of 25µmol photons m-2 s-1. Cultures were bubbled with sterile air containing 1% (v/v) CO2. Log phase cells (OD730nm=0.6) were harvested by centrifugation (2000×g for 12 min) washed once and then re-suspended in sulfate-free media (MgSO4 replaced by the same molarity of MgCl2). In addition, all S-containing trace metals in BG-11 were replaced by non-S containing metals. Cells were harvested and fixed for microarray analysis by adding 10% (v/v) ice-cold 5% phenol in ethanol stop solution at the following time points: before S-depravation (time 0, control), 1, 3, 6, 12, 24, 48 and 72 hr after S-depravation. S-deprivation with HEPES buffering control experiment was performed as described above, except that HEPES buffer was used upon sulfate removal. Bacterial samples for a time course were taken at time 0, 1, 12 and 24 hrs after sulfate withdrawal. Growth stage control experiment was done in parallel with S deprivation experiments. Samples were taken at 0, 1, 2.5, 4, 7, 11 and 48 hr after OD730nm reached 0.60. All the experiments were done in biological replicates.