The sRNA NsiR4 fine-tunes arginine synthesis in the cyanobacterium Synechocystis sp. PCC 6803 by post-transcriptional regulation of PirA

Abstract

As the only oxygenic phototrophs among prokaryotes, cyanobacteria employ intricate mechanisms to regulate common metabolic pathways. These mechanisms include small protein inhibitors exerting their function by protein–protein interaction with key metabolic enzymes and regulatory small RNAs (sRNAs). Here we show that the sRNA NsiR4, which is highly expressed under nitrogen limiting conditions, interacts with the mRNA of the recently described small protein PirA in the model strain Synechocystis sp. PCC 6803. In particular, NsiR4 targets the pirA 5ʹUTR close to the ribosome binding site. Heterologous reporter assays confirmed that this interaction interferes with pirA translation. PirA negatively impacts arginine synthesis under ammonium excess by competing with the central carbon/nitrogen regulator P II that binds to and thereby activates the key enzyme of arginine synthesis, N-acetyl-L-glutamate-kinase (NAGK). Consistently, ectopic nsiR4 expression in Synechocystis resulted in lowered PirA accumulation in response to ammonium upshifts, which also affected intracellular arginine pools. As NsiR4 and PirA are inversely regulated by the global nitrogen transcriptional regulator NtcA, this regulatory axis enables fine tuning of arginine synthesis and conveys additional metabolic flexibility under highly fluctuating nitrogen regimes. Pairs of small protein inhibitors and of sRNAs that control the abundance of these enzyme effectors at the post-transcriptional level appear as fundamental building blocks in the regulation of primary metabolism in cyanobacteria.

Diurnal rhythm causes metabolic crises in the cyanobacterial mutants of c-di-AMP signalling cascade

“…S6F ). The elevated PirA levels is in agreement with our transcriptome analysis revealing a low expression of the NtcA-dependent sRNA, NsiR4, the repressor of pirA (Bolay et al 2022), which further explains the low Arg levels in Δ dacA . The induction of IF17 is consistent with the increased levels of glutamine, since expression of gifB (encoding IF17) is mediated by a glutamine-dependent riboswitch (Klähn et al 2018).…”

Process Systems Engineering Approach for the Sustainable Design of Cyanobacteria-Based Integrated Biorefineries and Their Heat Exchanger Network

“…Also, it is important to note that the in silico strains S2 and S3 of Synechocystis are favored due to their superior biomass and ethanol productivities, emphasizing the significance of strain engineering for optimal performance in biorefineries. In the context of current research, gene editing and precise post-transcriptional control over gene expression tools, such as CRISPR-Cas9 − and small regulatory RNAs (sRNAs), − respectively, have emerged as revolutionary techniques for cyanobacteria strain design. This combined approach offers researchers unprecedented capabilities to engineer cyanobacteria with enhanced metabolic pathways, increased productivity, and specialized adaptability to varying environmental conditions.…”

Nitrite excretion by cyanobacteria is controlled by the small protein NirP1

“…NtcA or the PII regulator and proteins interacting with them [10][11][12][13][14][17][18][19]. However, unlike several other regulatory proteins that modulate the activity of other regulators (e.g., PirA and PipX), NirP1 targets one of the key enzymes of nitrogen assimilation metabolism directly.…”

“…Archetypical examples for these proteins are the inhibitory factors IF7 and IF17, as their binding to GS leads to the inactivation of the enzyme when nitrogen supply exceeds demand [17]. The PII-interacting regulator of arginine synthesis (PirA) regulates the flux into the ornithine-ammonia cycle and therefore arginine synthesis [18,19], while the PII-interacting regulator of carbon metabolism (PirC) functions as an inhibitor of 2,3-phosphoglycerate-independent phosphoglycerate mutase (PGAM), the enzyme that shifts newly fixed CO2 toward lower glycolysis [20]. PirC becomes sequestered by PII at low 2-OG levels and released for PGAM inhibition at high 2-OG levels and therefore connects the control of carbon and nitrogen assimilatory pathways in a particular way [20].…”