FIGURE 2.

GrlR represses the expression of locus of enterocyte effacement (LEE) genes in the absence of H-NS. (A) Expression of the LEE1-cat, LEE2-cat, LEE4-cat, and LEE5-cat fusions was analyzed in WT enteropathogenic Escherichia coli (EPEC) (black bars) and its Δhns isogenic mutant (white bars) carrying the empty vector pMPM-T3 (V) or its derivative pT3GrlR (R), grown in DMEM with shaking at 37°C. Specific chloramphenicol acetyltransferase (CAT) activity was determined using samples collected from cultures grown in 50 ml Dulbecco’s modified Eagle’s medium (DMEM) or Lysogeny Broth (LB) 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 < 0.01; ^***p-value < 0.001; ^****p-value < 0.0001). (B) Total extracts were prepared from the same culture samples and separated by 12% SDS-PAGE. The expression of Tir, intimin and EspA was analyzed by western blotting using polyclonal anti-intimin and anti-EspA and monoclonal anti-Tir antibodies. As controls for protein loading, maltose binding protein (MBP) and DnaK were also detected using monoclonal anti-DnaK and polyclonal anti-MBP antibodies.