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 substrain GT-T cultivated at ambient air or supplemented with 3% CO2. The acclimation to different CO2 concentrations is crucial for photoautotrophic organisms living in aquatic environments such as cyanobacteria. Samples were taken before and 1 h and 24 h after transfer to the3 % CO2 environment. The analyzed strains were wild type, a deletion mutant of gene ssl2982/rpoZ (ΔrpoZ) and two suppressor strains (R1, ΔrpoZ-S1 and R2, ΔrpoZ-S2). In cyanobacteria, elevated CO2 is known to down-regulate carbon concentrating mechanisms and accelerate photosynthesis and growth, but mechanism(s) of carbon signalling remains only poorly understood. Here we reveal a novel signalling cascade connecting the amount of CO2 and growth in the model cyanobacterium Synechocystis sp. PCC 6803. Deletion of the small ω subunit of the RNA polymerase (RNAP) in the ΔrpoZ strain prevents normal high-CO2-induced up-regulation of numerous photosynthetic genes, and low expression of peptidoglycan synthesis genes induced lysis of dividing ΔrpoZ cells in high CO2. Spontaneously raised secondary mutations in the ssr1600 gene rescued the high-CO2-sensitive phenotype of the ΔrpoZ strain. Biochemical analyses showed that the ssr1600 gene encodes an anti-σ factor antagonist of group 2 σ factor SigC, and 3D structural modelling suggest that Slr1861 functions as an anti-SigC factor. In ΔrpoZ, excess formation of RNAP-SigC lead to high CO2 sensitive phenotype, whereas the drastically reduced Ssr1600 content in the suppressor mutants reduce the formation of the RNAP-SigC holoenzyme to the similar level as in the control strain, allowing almost normal transcriptome and growth of suppressor lines in high CO2. We propose that the SigC σ factor, the anti-SigC factor Slr1861 and the anti-SigC antagonist Ssr1600 forms a growth regulating signalling cascade in cyanobacteria.
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 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 (ÎssaA) 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 ÎssaA 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 âssaA 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:Acclimation of cyanobacterium Synechocystis sp. PCC6803 to suboptimal conditions is largely dependent on adjustments of gene expression, which is highly controlled by the σ factor subunits of RNA polymerase (RNAP). The SigB and SigD σ factors are close homologues. Here we show that sigB and sigD genes are both induced in bright light and high temperature stresses. Comparison of transcriptomes of the control strain (CS), ΔsigB, ΔsigD, ΔsigBCE (SigD is an only functional group 2 σ factor), and ΔsigCDE (SigD is an only functional group 2 σ factor) strains in standard, bright light and high temperature conditions revealed that the SigB and SigD factors regulate different set of genes, and that SigB and SigD regulons are highly dependent on stress conditions. The SigB regulon is bigger than the SigD regulon at high temperature, whereas in bright light the SigD regulon is bigger the SigB regulon. Furthermore, our results show that favoring the SigB or SigD factor by deleting other group 2 σ factors do not lead to superior acclimation to bright light or high temperature conditions, indicating that all group 2 σ factors play roles in acclimation processes.