Fig. 3.

Arginine repressor ArgR is a direct activator of LEE gene expression in vitro. (A) qRT-PCR analysis to compare the expression of select virulence genes and (B) Western blot analysis of secreted proteins EspB and EspA from WT, ΔargR and cpl EHEC (complemented with argR gene on the pACYC184). Experiments in A and B were performed in microaerophilic conditions using low-glucose DMEM with arginine, and samples were harvested in late log phase. (C) For fluorescein actin staining analysis, HeLa cells were infected with WT, ΔargR and complemented argR EHEC (clp) and incubated in low-glucose DMEM with arginine for 4 h postinfection. Then, the cells were stained with FITC-phalloidin to visualize actin (green) and propidium iodide to stain for bacteria and nuclei (red). Pedestals were visualized as a green spot. Pedestals were enumerated for each field (60×). (D) The percentage of infected cells and the number of pedestals per infected cell were quantified (n = 3). (E) EMSA of His-tagged ArgR with argI promoter probe (positive control), kanamycin promoter (negative control), and segments of the EHEC LEE1 regulatory region numbered from the proximal transcriptional start site. Error bars represent SEs of means (SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.