Characterization of ail1 Promoter Binding

The promoter of ail1_Pl was PCR-amplified from the genomic DNA of TT01 strain using specific primers (Supplemental Table 1) and purified using the High Pure PCR Product Purification kit (ROCHE). The 5′ ends of DNA fragment were labeled using [γ-³²P] ATP and T4 polynucleotide kinase (Promega). Radioactive DNA probe (2000 cpm/ml), 200 ng of poly(dI-dC)-poly(dI-dC) (SIGMA) and different amounts of PhoP-His were mixed with binding buffer (50 mM tris-HCl pH 8, 50 mM KCl, 50 µg/ml BSA) in a total 20 µl volume and incubated for 20 min at room temperature. The mixture was then loaded onto a native 6% (w/v) polyacrylamide TBE precast Gel (Invitrogen) and electrophoresed in 1% TBE (Tris-Borate-EDTA) buffer for 1 h at 100 V. Radioactive species were detected by autoradiography. PhoP-His was activated by in vitro phosphorylation with acetyl phosphate as previously described [49] (link).