Electrophoretic mobility shift assays with NanR and probes covering the nanA-nagA intergenic region. DNA probes containing the nagA-nanA intergenic region were incubated with various concentrations of NanR. (A) Schematic illustration of the intergenic region of the C. glutamicum
nagA and nanA genes, probes used for EMSAs, and localization of NanR-binding site. (B and C) Representative EMSAs using NanR (0, 0.1, 0.2, and 0.3 μg) with 10 ng of the probes PnagA (B) and PnagA (C). (D) EMSAs using 0.3 μg of NanR (indicated by plus signs) with 10 ng of the probes PnagA-A or PnagA-B. (E) Identification of GlcNAc-6P and ManNAc-6P as NanR effector molecules. Shown are results of EMSAs using NanR (0.2 μg; 0.7 μM) with the Probe PnagA (10 ng; 3.7 nM) and 0.5, 5, or 50 μg of GlcNAc-6P (0.17 mM, 1.66 mM, and 16.6 mM) or 0.5, 5, or 50 μg of ManNAc-6P (0.14 mM, 1.44 mM, and 14.49 mM). Lanes M, molecular size markers.