FIGURE 3.

GrlR is the main repressor of locus of enterocyte effacement (LEE) gene expression under repressing conditions. (A) Expression of the LEE1-cat, LEE2-cat and LEE5-cat fusions was analyzed in WT EPEC (black bars) and its ΔgrlR (gray bars) and Δhns (white bars) isogenic mutants, grown in 50 ml of Dulbecco’s modified Eagle’s medium (DMEM) or Lysogeny Broth (LB) with shaking at 37°C. Specific chloramphenicol acetyltransferase (CAT) activity was determined from samples collected from cultures at an OD₆₀₀ of 1. Values are an average of three independent experiments performed in duplicate. Error bars indicate standard deviations. Statistically different values are indicated (^****p-value < 0.0001). (B) Secreted proteins of the same cultures were concentrated from supernatants by precipitation with trichloroacetic acid (TCA), separated by 12% SDS-PAGE and stained with Coomassie brilliant blue.