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

Significance A key enzyme in inorganic nitrogen assimilation, glutamine synthetase, is the target of multiple regulatory mechanisms. Here we describe the nitrogen stress-induced RNA 4 (NsiR4), a small regulatory RNA that reduces the expression of inactivating factor 7 (IF7), an inhibitory factor of glutamine synthetase in cyanobacteria. The expression of NsiR4 is under positive control through the nitrogen control transcription factor (NtcA). NtcA also induces the transcription of the glutamine synthetase gene and represses the gene encoding IF7. Therefore, NsiR4 is a new player in the NtcA-mediated regulation of nitrogen assimilation, which is important for adaptations to rapid changes in available nitrogen sources and concentrations. 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 on 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 (IF)7. 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 GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward 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, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.

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