OxyS represses mepS expression. (A) Nucleotides 50–84 in OxyS (marked in green) are involved in nusG binding, while nucleotides 25–33 (purple) bind mepS. In OxyS, Δ15–33 mutant nts 15–33 were deleted. In OxyS C18G G30C, the mutation G30C complements (-18)G in mepS, while C18G restores the disrupted OxyS structure. (B) Predicted base-pairing between mepS (red) and OxyS (purple) RNAs. Indicated are the initiation codon (bold) and the Shine–Dalgarno sequence of mepS (underlined). The position of mepS and OxyS complementary mutations is indicated. (C,D) Cultures (MG1655 mal::lacIq ΔlacZ::Tn10) carrying mepS-lacZ wild-type and mepS C(-18)G-lacZ (pSC101*) translational fusions and Plac-OxyS wild-type and mutants were treated with IPTG (1 mM) at OD600 of 0.1–0.2. β-Galactosidase activity was measured 60 min after treatment. Results are displayed as mean of three to six biological experiments ± standard deviation. (E) To detect the effect of OxyS on MepS protein levels, the sequential peptide affinity (SPA) tag was inserted into the MG1655 (mal::lacIq) chromosome adjacent to the carboxy-terminal amino acid of MepS. At OD600 of 0.5, the cultures were exposed to 1 mM H2O2 for the indicated time points, whereas cultures carrying OxyS plasmids were treated with 1 mM IPTG for 30 min to induce OxyS expression. Plasmid-encoded (see Fig. 2E, wild-type) and H2O2-induced (Fig. 1E, northern) OxyS RNA levels were detected using end-labeled OxyS-specific primer (3708). Loading control (tm RNA) was detected using primer 1912.
