Mlc directly represses hilE by binding to the P3 promoter 
A gel mobility shift assay was performed with purified Mlc, to test whether Mlc directly represses the hilE P3 promoter.
The Mlc-His6 protein was produced in E. coli and purified to >90% homogeneity by Ni2+ affinity chromatography.
The activity of the purified Mlc-His6 protein was verified with the control gel mobility shift experiment employing the Salmonella ptsG DNA fragment that contains the known Mlc-binding site (data not shown).
When the labeled hilE promoter DNA was incubated with 2-fold dilutions of the purified Mlc-His6 protein (100-800nM) in the presence of the non-specific DNA competitor poly dI-dC, the concentration-dependent formation of protein-DNA complexes was observed (Figure 5A).
The addition of cold probe released the labeled probe from the retarded complex (Figure 5A, right panel), which indicates specific binding of Mlc to the hilE P3 promoter DNA.
The precise locations of the Mlc-binding sites were determined by DNase I footprinting with His-tagged Mlc.
Two Mlc-binding sites, one at position -10 to +12 (Mlc 1) and the other at position -86 to -108 (Mlc 2) with respect to the transcriptional start site of hilE P3, were identified (Figure 5B).
Inspection of the DNA sequences at these sites showed high-level homology with the known consensus Mlc-binding sequence, which has the conserved TT-9bp-AA motif and an AT-rich region at positions +/-7 to +/-11, showing imperfect dyad symmetry (38) (Figure 5C).
These results clearly demonstrate that Mlc can regulate directly the hilE P3 promoter by binding to the promoter.
