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 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 NH + 4 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.regulatory RNA | Synechocystis | nitrogen assimilation | glutamine synthetase inactivating factors | NtcA

Chlorosis as a Developmental Program in Cyanobacteria: The Proteomic Fundament for Survival and Awakening

“…Therefore, the inhibitory proteins that regulate the glutamine synthetase enzyme, IF7 and IF17, must be tuned down. Accordingly, repression of the corresponding gif genes (ssl1911 and sll1515) is further enhanced by antisense RNAs (11,53), and we observed the same trend at the protein level: IF7 levels remained at their lowest until cells reached the vegetative state and IF17 levels showed an increase at T24, which indicated the first signs of nitrogen replenishment. Conversely to the levels of the glutamine synthetase inhibitory proteins, the levels of the nitrogen-starvation-specific glutamine synthetase GlnN (Slr0288) (54,55) were threefold higher in chlorotic cells and lower after completion of recovery.…”

Chlorosis as a developmental program in cyanobacteria: the proteomic fundament for survival and awakening

“…Therefore, the inhibitory proteins that 703 regulate the glutamine synthetase enzyme, IF7 and IF17, must be tuned down. 704 Accordingly, repression of the corresponding gif genes (ssl1911 and sll1515) is further 705 enhanced by antisense RNAs (11,53), and we observed the same trend at the protein 706 level: IF7 levels remained at their lowest until cells reached the vegetative state and 707 IF17 levels showed an increase at T24, which indicated the first signs of nitrogen 708 replenishment. Conversely to the levels of the glutamine synthetase inhibitory proteins, 709 the levels of the nitrogen-starvation-specific glutamine synthetase GlnN (Slr0288) 710 (54,55) were threefold higher in chlorotic cells and lower after completion of recovery.…”

Elements of the heterocyst‐specific transcriptome unravelled by co‐expression analysis in Nostoc sp. PCC 7120

“…Both trans-acting sRNAs and antisense RNAs have been described as regulators of cyanobacterial metabolism, including acclimation to changing iron availability (Georg et al, 2017), carbon supply (Eisenhut et al, 2012) or light intensity (Georg et al, 2014). Nitrogen-regulated sRNAs are involved in feed-forward regulatory loops providing additional levels of response to nitrogen deficiency (Klähn et al, 2015;Alvarez-Escribano et al, 2018). Similarly, heterocystspecific sRNAs (NsiR1, NsiR2, NsiR8 or NsiR9) and antisense transcripts identified in the E-DIF and L-DIF groups of transcriptional responses could modulate the stability and/or translation of their corresponding target transcripts specifically in developing heterocysts, thus contributing to the metabolic rearrangements that take place in these specialized cells (Olmedo-Verd et al, 2019).…”