FIGURE 6.

GrlA antagonizes GrlR-mediated repression. (A) Profile of secreted proteins from WT enteropathogenic Escherichia coli (EPEC) carrying the empty vector pMPM-T3 or its derivatives pT3GrlR, pTEPGrlA1, and pT3GrlRA, pT3GrlRA/I44A and pT3GrlRA/R54A grown in 50 ml Dulbecco’s modified Eagle’s medium (DMEM) or Lysogeny Broth (LB) to an OD₆₀₀ of 1. Secreted proteins were concentrated from culture supernatants by precipitation with trichloroacetic acid (TCA), separated by 12% SDS-PAGE and stained with Coomassie brilliant blue. (B) Secreted proteins from EPEC ΔgrlR carrying the same plasmids as in panel (A) grown in 50 ml of LB with shaking at 37°C. (C) Total extracts were prepared from bacterial samples of the same cultures described in panel (B). Proteins were separated by 12% SDS-PAGE and expression of intimin, Tir and EspA was analyzed by western blotting using anti-intimin, anti-Tir and anti-EspA antibodies. As a control for protein loading, DnaK expression was also analyzed using anti-DnaK antibodies.