Project description:Unicellular cyanobacteria that do not fix nitrogen can survive prolonged periods of nitrogen starvation as bleached cells in a non-growing, dormant state. Upon re-addition of a usable nitrogen source, bleached cultures re-green within 48 hours and the cells return to vegetative growth. Here we investigated the process of resuscitation at the physiological and molecular level. Almost immediately upon nitrate addition, the cells initiate an amazingly organized resuscitation program: they first turn on respiration, gaining energy and activating the genes of the entire translational apparatus, genes for ATP synthesis and nitrate assimilation. Only after about 12 hours, the cells rebuild the photosynthetic apparatus and switch on photosynthesis. Analysis of the transcriptome in recovering cells shows a perfect match to the physiological processes and reveals a paramount dynamics of non-coding RNAs in awaking cells. This genetically encoded program ensures rapid colonization of habitats, in which nitrogen starvation imposes a recurring growth limitation.
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:Unicellular cyanobacteria that do not fix nitrogen can survive prolonged periods of nitrogen starvation as bleached cells in a non-growing, dormant state. Upon re-addition of a usable nitrogen source, bleached cultures re-green within 48 hours and the cells return to vegetative growth. Here we investigated the process of resuscitation at the physiological and molecular level. Almost immediately upon nitrate addition, the cells initiate an amazingly organized resuscitation program: they first turn on respiration, gaining energy and activating the genes of the entire translational apparatus, genes for ATP synthesis and nitrate assimilation. Only after about 12 hours, the cells rebuild the photosynthetic apparatus and switch on photosynthesis. Analysis of the transcriptome in recovering cells shows a perfect match to the physiological processes and reveals a paramount dynamics of non-coding RNAs in awaking cells. This genetically encoded program ensures rapid colonization of habitats, in which nitrogen starvation imposes a recurring growth limitation. Synechocstis PCC 6803 WT cells were subjected to nitrogen limitation for 14d, then nitrogen was re-added to monitor recovery of the cells. Samples were taken before nitrogen depletion, after 14d of nitrogen depletion and 4h, 13h, 24h and 48h after nitrogen re-addition. Samples were taken in biological replicates for all timepoints besides 48h nitrogen recovery.
Project description:Small proteins are an underinvestigated class of gene products in all domains of life. Here we describe the role of NsiR6/NblD, a cysteine-rich 66 amino acid small protein in the acclimation response of cyanobacteria to nitrogen starvation. Phycobilisomes, the macromolecular pigment-protein complexes for photosynthetic light harvesting, are rapidly degraded upon shift to low nitrogen. Deletion of nblD in Synechocystis sp. strain PCC 6803 prevents this degradation, indicated by the non-bleaching (nbl) phenotype. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type, while overexpression of NblD under nitrogen-replete conditions did not lead to any phenotypical effect, different from the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. However, transcriptome analysis revealed that nitrogen starvation induced nblA1/2 transcription in the ΔnblD strain, which excluded the possibility that the nbl phenotype was due to a possible NblD function as transcriptional co-regulator. In contrast, fractionation experiments indicated the presence of NblD in the phycobilisome fraction and pull-down experiments with NblD containing a triple FLAG tag identified the α and β phycocyanin subunits as the only two co-purifying proteins. Homologs of NblD exist in all cyanobacteria that use phycobilisomes but not in the genera Prochlorococcus and Acaryochloris which use alternative light-harvesting mechanisms. These data suggest that NblD plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting. We performed a microarray, to examine the global expression pattern of wild type and ∆nblD isolated RNA after 0h and 3h past induction of nitrogen depletion (-N) to detect potential differences in the nitrogen acclimation process.
Project description:The protein abundance of Synechocystis in response to the given copper-iron combination was examined, then followed by comparative proteomic analyses to reveal the proteins associated with copper and iron stress. These data would provide a theoretical basis for understanding the relationship between copper and iron in cyanobacteria at the protein level and shed light on the role of these two metal elements in energy metabolism and biomass accumulation of cyanobacteria.
Project description:The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7
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.
Project description:Background: The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some 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. Results: 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 are accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in ΔssaA. In vivo pull-down studies of the RNA polymerase complex indicated that the presence of 6S RNA promotes the recruitment of the cyanobacterial housekeeping σ factor SigA, concurrently supporting dissociation of group 2 σ factors during recovery from nitrogen starvation. Conclusions: The combination of genetic, physiological and biochemical studies reveals the homologue of 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by accelerating the switch from group 2 σ factors SigB, SigC and SigE to SigA-dependent transcription. We therefore introduce the cyanobacterial 6S RNA as a novel candidate regulator of RNA polymerase sigma factor recruitment in Synechocystis sp. PCC 6803. Further studies on mechanistic features of the postulated interaction should shed additional light on the complexity of transcriptional regulation in cyanobacteria.
Project description:Gene expression changes were followed in cultures of the cyanobacterium Synechocystis sp. PCC 6803 three and 24 hours after shift from high carbon (HC) to low carbon (LC) concentrations. The acclimation to fluctuating inorganic carbon (Ci) concentrations is crucial for photoautotrophic organisms living in aquatic environments such as cyanobacteria. The PII-like regulator protein SbtB binds the second messengers cAMP or c-diAMP and is involved in the acclimation to LC. Here, we investigated the impact of SbtB and of second messengers on gene expression changes during this acclimation response. We analyzed cultures of the wild type (WT), the ΔsbtB mutant lacking the PII-like regulator protein SbtB, the ΔcyaI mutant, lacking the main soluble adenylate cyclase for cAMP production and ΔdacA, lacking the diadenylate cyclase for c-diAMP production. The majority of LC-induced genes behaved in these mutants like in wild type. However, a defined subset of LC-regulated genes in WT was found to be changed in mutant ΔsbtB already under high CO2. Collectively, the results indicate that SbtB regulates a particular subset of genes during the LC acclimation response.