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 5'UTR 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.

Keywords: Cyanobacteria; RNA regulator; arginine metabolism; nitrogen assimilation; posttranscriptional regulation; sRNA.

Figure 1.

In vivo reporter assays for the verification of direct interaction between the 5ʹUTR of pirA and NsiR4. A: Computational interaction prediction between the 5ʹUTR of pirA and NsiR4 using the IntaRNA tool [54,55]. The numbers refer to the TSS +1. The start codon of pirA is highlighted in green while inserted mutations are highlighted in red. B: GFP fluorescence in E. coli TOP 10 strains with different plasmid combinations expressing NsiR4 or a nonsense RNA (plasmid pJV300) in presence of the 5’UTR of pirA which was fused to a sfgfp gene, as well as a negative control accounting for the autofluorescence of the cells (pXG0). It should be noted that the mutation, which was introduced into the pirA 5ʹUTR affected translation and hence, the GFP fluorescence detected for these constructs (column 5–6) was generally lower as observed for the WT version. Data are the mean ± SD of two independent experiments each using six independent cultures/clones. Asterisks label statistically different mean values, confirmed by single factor analysis of variance (ANOVA).

Figure 2.

PirA accumulation kinetics in different NsiR4 mutant strains. A: Representative Western blots using antibodies specific against PirA [32]. Equal loading is confirmed by the simultaneous detection of thioredoxin (TrxA) using specific TrxA antibodies. At time point 0, PirA accumulation was stimulated by the addition of 10 mM ammonium (provided as ammonium chloride) to cells that were pre-cultivated in BG11 supplemented with nitrate as sole N source. Prior to sampling all cultures were supplemented with 1 µM CuSO₄ and further cultivated for 12 hours to ensure sufficient overexpression of NsiR4 in strains NsiR4oex and ΔnsiR4::oex as described previously [40]. B: Densitometric analysis of the obtained signal intensity for PirA using ImageJ software. All values were normalized to the corresponding signal for TrxA of the same blot and time point. The data are shown as relative intensities compared to the signal for the WT at 360 min (set as 1).

Figure 3.

Implications of enhanced NsiR4 accumulation on arginine metabolism. Left panel: Shown are arginine accumulation kinetics in WT and strains ΔnsiR4 and NsiR4oex in response to addition of 10 mM ammonium. Data are the mean ± SD of three independent replicates. Asterisks label significant deviation from the WT controls, confirmed by single factor analysis of variance (ANOVA). Right panel: Schematic of the effects of the NsiR4-PirA module on arginine synthesis by modulating NAGK activity before and after ammonium shock.

Figure 4.

The current status of the regulatory network of NsiR4 in Synechocystis. A: Schematic overview of the NtcA-NsiR4-IF7-PirA network and its impact on N metabolism. The network has been built based on data from this study and previous work [25,32,40]. B: Proposed model of the NtcA-NsiR4 feed-forward loop and its effect on the accumulation of corresponding gene products in response to fluctuating N supply (data based on [25,32,40,65,67,72]). The solid line represents target protein expression in a system omitting NsiR4, the dotted line describes a system including NsiR4. C: Regulatory relations between the different players of the NtcA-NsiR4-IF7-PirA network resulting in a multi-output AND-gated type 3 coherent feed-forward loop with a reversed sign-sensitive behaviour [65].