Differential regulation of Escherichia coli topoisomerase I by Fis


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We previously reported that the P1 promoter of topA encoding topoisomerase I of Escherichia coli is activated in response to oxidative stress, in a Fis-dependent manner. Here we show that Fis regulation of topA varies with the intracellular concentrations of Fis. Thus, when Fis levels are low, hydrogen peroxide treatment results in topA activation, whereas at high Fis levels hydrogen peroxide treatment renders topA P1 inactive. In vivo DMS footprinting indicates that only at low Fis levels, when exposed to the stress, the region of the topA promoter changes and P1 becomes active. Potassium permanganate experiments indicate that low levels of Fis activate P1 transcription by facilitating the formation of open complexes, while high levels of this protein shut off the promoter. DNase I footprinting show that Fis binds the promoter region of topA at eight sites with different affinities. One low affinity site overlaps the −10, −35 hexamers of RNA polymerase. We propose that in response to oxidative stress, when present at low levels, Fis binds the promoter region of topA at its high affinity sites, thereby facilitating the recruitment of RNA polymerase to P1, while at high levels, Fis occupies the low affinity sites as well, and thus prevents the binding of RNA polymerase. Our results indicate that the oxidative stress response varies in response to changes in growth phase and nutritional environment.