Fig. 6. MgrR controls cesT expression.

(A) Network of MgrR interactions with accessory genome–encoded genes, revealed by RIL-seq. Network annotation is as in Fig. 5. (B) Western blots of Tir, CesT, and intimin extracted from wild-type EPEC (wt), Δhfq mutant, and EPEC carrying either a plasmid encoding mgrR under the inducible Tet promoter (pZA11-mgrR) or a plasmid constitutively expressing mgrR (pZE12-mgrR), grown under the activating condition. Where indicated, anhydrotetracycline (aTc) was used to induce MgrR expression. Total protein was used as a loading control. Representative blots of three independent experiments. (C and D) EPEC strains containing cesT-gfp transcriptional (C) or translational (D) fusion, transformed with either pZA11-mgrR or pZE12-mgrR plasmids, were grown under activating condition and analyzed for GFP fluorescence intensity. The genetic context of the cesT-gfp fused gene is displayed above the plot, with different genes represented as arrows. Means of three biological repeats are displayed by black lines, while individual repeats are shown (green dots). Error bars signify SEs. Asterisks denote statistically significantly lower fluorescence compared to the control (P ≤ 0.05, Wilcoxon rank sum test, one-tailed). a.u., arbitrary units. (E) IntaRNA (39) predicted base pairing of cesT 5′UTR region and MgrR. In red are mutated nucleotides for the cesT* and MgrR* mutations. LEE5 genes are depicted by arrows. (F) Western blots of CesT-3xFlag from wild-type (wt) and cesT* mutant strains, carrying plasmids expressing wild-type MgrR or MgrR* mutant (pZE12-mgrR/mgrR*), grown under the activating condition. Where indicated, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to induce expression of MgrR or MgrR*. Representative blots of three independent experiments.