Characterization of putative BvgA binding sitesinside the fhaB upstream region of B. holmesii. (a) Schematic representation of the fhaB upstream region containing the four putative BvgA binding sites BS1–BS4 and representation of the PCR amplified DNA probes I-V used for band shift experiments with the purified B. holmesii BvgABH protein. Numbers indicate the distance from the Bvg-dependent fhaB transcriptional start site (P3) taken as position +1. (b) Binding of the B. holmesii BvgABH protein to DNA probes I-V, containing different amounts of putative BvgA binding sites (a); the radiolabelled PCR fragments were incubated with 150 ng (lane 2), 300 ng (lanes 3 and 8), 400 ng (lanes 4 and 9), 500 ng (lanes 5, 10, 13, 17 and 21), 600 ng (lane 22), 700 ng (lanes 6, 11, 14, 18 and 23) and 800 ng (lanes 15, 19 and 24) of in vitro phosphorylated BvgABH. No protein was added in lane 1 (DNA probe I), lane 7 (DNA probe II), lane 12 (DNA probe III), lane 16 (DNA probe IV) and lane 20 (DNA probe V). The reaction mixtures were run on a non-denaturating 4% polyacrylamide gel. F, free DNA probes; C, DNA-protein complexes.