FIG. 3.

DNA gel shift experiments to examine the effects of different mutations on the binding of the 52-bp fragment to the partially purified PtxS. The lysate of the E. coli strain K38/pJAC17 was used as a source of the PtxS protein as previously described (15). We used the 38-bp deletion fragment and the 52-bp fragments that carried different mutations that were synthesized from pBS9 or its derivatives by PCR in the DNA gel shift assay as described in the text. Each DNA binding mixture contained 10⁵ cpm of the labeled 52-bp fragment and approximately 2 μg of the cell lysate. With the exception of the reaction mixture with pBS9Δ, all DNA binding reaction mixtures contained the 52-bp fragments that were obtained from their respective plasmids. The binding reaction mixture with pBS9Δ contained the 38-bp deletion fragment. In the control lane, the 50-bp fragment was incubated with the lysate of the E. coli strain K38/pT7-5 only. −, the DNA binding reaction mixtures containing the probe only; +, the DNA binding reaction mixtures in which the probe was incubated with cell lysate. The faint band in pBS9Δ was not detected in several repeated experiments (data not shown). Similar faint bands that migrated at the same lower distance were also detected in pBS9A5/5′ and pBS9C7 (in some but not all experiments), even though these reaction mixtures contained the 52-bp fragment and not the 38-bp deletion fragment.