To visualize binding of FabR protein to the two target genes, fabA and fabB, gel shift experiments were employed (Feng and Cronan, 2009b (link), 2010 (link)) with minor improvements. The E. coli FabR binding palindromes were synthesized using two complementary oligonucleotides (fabAec_P-F plus fabAec_P-R for fabA gene and fabBec_P-F plus fabBec_P-R for fabB gene) (Table 2). The V. cholerae fabA probe covering a predicted FabR binding site was PCR amplified with two primers, fabAec_P-F plus fabAec_P-R, and a DNA fragment containing fabB promoter region (257 bp) was obtained with a pair of primers, fabBec_P-F plus fabBec_P-R (Table 2). The probes were digoxigenin-labeled by terminal transferase incorporation of digoxigenin-ddUTP (Roche). For gel shift assays the digoxigenin-labeled DNA probes (~ 0.1 pmol) were mixed with serial dilutions of the in vitro translated FabR protein in binding buffer (20 μl total volume) (Roche) at room temperature for 15 min. The DNA/protein mixtures were loaded onto 7% native PAGE and after running the gel contents were transferred to an equilibrated, positively charged nylon membrane (Roche) by contact blotting. The luminescence signal was developed in CSPD working solution at room temperature following the incubation of the nylon membrane with an anti-digoxigenin antibody. Two hours later an exposure of the nylon membrane to an enhanced chemiluminescence (ECL, Amersham) detection system gave signal capture.