EMSA was performed using a LightShift™ EMSA Optimization & Control Kit (Thermo-Fisher Scientific, Waltham, MA) with biotin-labeled DNA probes and purified NigR according to the manufacturer’s instructions. Poly(dI-dC) was used in all reactions as a non-specific competitor at a concentration of 1 μg per reaction (Ajdic & Ferretti, 1998 (link); Nieto et al., 2001 (link); Chawla et al., 2010 (link)). Unlabeled specific competitor was added where indicated. Both non-specific and specific competitors were used as described previously (Ajdic & Ferretti, 1998 (link); Gaigalat et al., 2007 (link); Nentwich et al., 2009 (link)). An unrelated protein InvB (Type III secretion chaperone from Salmonella enterica; Lilic et al., 2006 (link)) was used as a negative control. The 20-μl binding reactions were incubated at room temperature for 20 min and the DNA–protein complex was separated from the free probe by electrophoresis on 4 or 5% native polyacrylamide gel in TBE buffer. The material was transferred to positively charged Hybond nylon membrane (GE Healthcare, Pittsburgh, PA) using Trans-Blot Semi Dry Transfer Cell (Bio-Rad) according to the manufacturer’s instructions. The membrane was cross-linked for 10 min using UV Crosslinker (UVP HL-200 HybriLinker) and developed using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) following the manufacturer’s instructions.