DNA Shift Assays of LdtR Mutants

DNA shift assays of LdtR, as well as its mutated versions, was carried out as described previously [9 (link)]. Fragments of the selected promoters were generated by PCR using biotinylated primers (Table 2). The reaction mix for EMSA contained 1 ng of 5’biotin-labelled DNA probe, 50 nM Tris-HCl pH 7.2, 150 mM KCl, 10 mM MgCl₂, 0.01% Triton X100, 12.5 ng/μl both Poly(dI-dC) and Poly(dA-dT) nonspecific competitor DNAs, and purified LdtR (0–1000 nM) as indicated. The mix was incubated for 20 min at 37°C and the electrophoresis was conducted at 4°C on 6% acrylamide/bisacrylamide non-denaturing gels in 0.5X Tris-borate EDTA buffer (TBE) pH 8.3. The DNA was transferred to a Hybond-N⁺ membrane (GE Healthcare, Pittsburgh, PA, USA) by electroblotting at 250 mA for 45 min in a Semi Dry Electroblotting System (Fisher Scientific). The detection of the DNA bands was carried out with the Phototope-Star Detection Kit (New England Biolabs, Ipswich, MA, USA). The membranes were exposed to Autoradiography Film (Mid-Sci, St. Louis, MI, USA).