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The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7

ABSTRACT: Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen-limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling upon pulse overexpression and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5’UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor IF7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus NH4+ assimilation via glutamine synthetase. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between wild type and an NsiR4 knock-out mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis towards oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.

ORGANISM(S): Synechocystis sp. PCC 6803

PROVIDER: GSE73840 | GEO | 2015/10/20

SECONDARY ACCESSION(S): PRJNA298210

REPOSITORIES: GEO

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Project description:Unlike all other bacteria, the physiology of cyanobacteria is based on the assimilation of organic matter from inorganic carbon driven by oxygenic photosynthesis. This setting poses special regulatory challenges, particularly in integrating carbon and nitrogen metabolism. Several regulatory factors control the primary nitrogen metabolism, such as the PII protein and proteins interacting with it (PirA, PirC or PipX), or the inactivating factors IF7 and IF17 impacting glutamine synthetase activity. However, regulatory proteins of other enzymes in the nitrogen assimilation pathway have not been described thus far. Here, we show that the 81 amino acids regulatory protein NirP1 in the cyanobacterium Synechocystis sp. PCC 6803 functions as an inhibitor of nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Ectopic overexpression of the nirP1 gene under standard growth conditions led to a pigmentation phenotype and delayed growth, which was correlated by the excretion of nitrite and dramatic changes in metabolite pools. We show that nitrite excretion occurs in Synechocystis wild type cells upon the shift from high CO2 (HC) to low CO2 (LC) conditions when NirP1 expression becomes activated. The expression of nirP1 is controlled by two transcription factors, NtcA, which binds to a recognition site in a repressive position above the transcription start site (TSS), and an activator, which probably binds to an element 60 to 43 nt upstream of the TSS. Therefore, the joint activity of NtcA and this activator leads to the observed induction of NirP1 under LC conditions, whereas it is repressed by shifts to nitrogen starvation. The data demonstrate that NirP1 plays a crucial role in the coordination of C and N primary metabolism in cyanobacteria by targeting the activity of one of the central enzymes. In natural environments, the excreted nitrite will be utilized by other microorganisms; therefore, NirP1 will ultimately impact the activities and composition of the surrounding microbiome.

Project description:Iron is an essential cofactor in many metabolic reactions. Mechanisms controlling iron homeostasis need to respond to changes in extracellular conditions, but must also keep the concentration of intracellular iron under strict control, as free ferrous iron (Fe2+) can lead to the generation of reactive oxygen species. Due to its role as redox carrier in photosynthesis, the iron quota in cyanobacteria is about 10 times higher than in model enterobacteria, but the molecular details how such high quota is tightly regulated have remained obscure. Here, we measured time-resolved gene expression changes after iron depletion in the cyanobacterium Synechocystis sp. PCC 6803 using a comprehensive microarray platform monitoring both protein-coding and non-coding transcripts. In total, 644 protein-coding genes were differentially expressed during the first 72h. Many of these proteins are associated with iron transport, photosynthesis or ATP synthesis. Comparing our data with three previous studies, we identified a core set of 28 genes involved in the iron stress response. Among them were genes encoding proteins important for assimilation of inorganic carbon, suggesting a previously unknown link between the carbon and iron regulatory networks. Nine of the 28 genes are of unknown function and constitute key targets for detailed functional analysis. Applying identical statistical and clustering criteria as for the protein-coding fraction, we also identified 10 small RNAs, 62 anti-sense RNAs, four 5M-bM-^@M-^YUTRs and 7 intergenic elements as likely to be involved in the iron regulatory network. Hence, our genome-wide profiling results indicate an unprecedented complexity in the iron-related regulatory network of cyanobacteria. We monitored iron limitation stress induced gene expression changes in the cyanobacterial model organism Synechocystis sp. PCC 6803. We included a control sample without iron-stress (0h) and 5 samples from timepoints after stress induction (3h, 12h, 24h, 48h, 72h). Each timepoint was sampled from two independent biological replicates.

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The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7

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