Figure 9.

EbfC and BpaB compete for binding to erp Operator 2. (A) EMSAs in which the concentration of BpaB321 was held constant while increasing concentrations of EbfC were added. (Lane 1) Labeled probe b-WT alone. (Lanes 2 and 3) Probe b-WT preincubated with 2.05 and 2.89 µM recombinant EbfC or BpaB321, respectively. (Lanes 4–12) Probe b-WT preincubated with 0.97 µM recombinant BpaB321, plus EbfC at concentrations of 0.27, 0.51, 1.03, 1.54, 2.05, 2.56, 3.08, 3.59 and 4.10 µM, respectively. Compositions of EMSA bands are indicated to the right of the panel. Asterisks indicate nonspecific DNA binding by EbfC observed at high concentrations of that protein (our unpublished data). Ratios of free DNA to BpaB–DNA complex 1 in lanes 4 through 12 were 3.9 : 1, 3.5 : 1, 2.7 : 1, 2.1 : 1, 1.4 : 1, 1.1 : 1, 1.1 : 1, 0.86 : 1 and 0.57 : 1, respectively, with the BpaB–DNA complex signals remaining constant, indicating that EbfC preferentially bound to free DNA. Quantification of signal intensities indicated that the proportion of EbfC–DNA complexes steadily increased with addition of EbfC protein, and that total of signal strengths remained constant across the EMSA gel. (B) EMSAs in which the concentration of EbfC was held constant while increasing concentrations of BpaB321 were added. (Lane 1) Labeled probe b-WT alone. (Lanes 2 and 3) Probe b-WT pre-incubated with 0.2 and 0.3 µM recombinant EbfC or BpaB321, respectively. (Lanes 4–12) Probe b-WT preincubated with 0.3 µM recombinant BpaB321, plus EbfC at concentrations of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 µM, respectively. Compositions of EMSA bands are indicated to the right of the panel. The asterisk indicates nonspecific DNA binding by EbfC observed at high concentrations of that protein (our unpublished data). Quantification of signal strengths indicated that proportions of free DNA and DNA bound by EbfC decreased as additional BpaB was added, while proportions of BpaB–DNA complexes increased.