Figure 5.

Stress-dependent mRNA editing dynamics are largely independent of changes in tadA expression. (A) Changes in tadA abundance in response to 0.5 mM or 1.0 mM H₂O₂ at different time points post exposure in S. pyogenes SF370. gyrA, rpoB and era served as reference genes for qRT-PCR, and tadA expression was normalised to the mean expression at t = 0 min. Statistical analysis was performed using two-way ANOVA and Dunnett's post-hoc test. (B) Schematic overview of the tadA locus and the integration of the AHT-inducible P_tet cassette. The cassette harbouring the antibiotic marker cat86, the repressor tetR and the promoter with three operator sites (P_tet) was inserted downstream of the SPy_0208-tadA operon (‘native’ tadA expression, EC3570) or upstream of tadA (‘AHT-inducible’ tadA expression, EC3622). (C, D) Effect of H₂O₂, zinc and AHT exposure on tadA abundance (left panel) and pepN editing (right panel) under native (C) and inducible tadA expression conditions (D). Strains in exponential phase were treated for 30 min with water (control, ‘ctrl’), 1 mM H₂O₂, 0.5 mM ZnSO₄ or 100 ng/ml AHT. tadA abundance was measured by qRT-PCR with gyrA, rpoB and era as reference genes, and pepN editing levels were examined by Sanger sequencing. Expression and editing levels were normalised relative to the control condition for each strain. Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test. *** P < 0.001; ** P < 0.01; * P < 0.05; ns, not significant.