FIGURE 7.

Influence of IHF consensus site mutations on tssD2a promoter activity and IHF binding. (A) The ptssD2aM-lux was constructed by introducing 4-bp changes in the IHF consensus site at the tssD2a promoter region. (B) Overnight cultures of V. fluvialis WT and ΔihfA strains containing either ptssD2a-lux or ptssD2aM-lux reporter fusions were diluted 1:100 in LB medium and 200 μl aliquots were transferred to Opaque-wall 96-well microtiter plates. The plates were incubated at 30°C with shaking for measuring the OD₆₀₀ and light units. Luminescence activity is calculated as light units/OD₆₀₀. The data represent three independent experiments. ^∗∗Significantly different between the ptssD2a-lux and the ptssD2aM-lux reporter fusions (t-test, P < 0.01). (C) EMSAs for IHF binding to wild-type tssD2_a promoter (left, probe-tssD2a) or to its mutations (right, probe-tssD2aM). Assays were performed as described in the Section “Materials and Methods.” The biotin-labeled 375-bp DNA probes (20 ng) was incubated with increasing amounts of purified V. cholerae IHF protein. The arrow on the left side indicates the unbound free probe, whereas the arrow on the right side indicates the probe bound to IHF protein.