Computation-directed identification of OxyR DNA binding sites in Escherichia coli

A computational search was carried out to identify additional targets for t he Escherichia coli OxyR transcription factor, This approach predicted OxyR binding sites upstream of dsbG, encoding a periplasmic disulfide bond chap erone-isomerase; upstream of fhuF, encoding a protein required for iron upt ake; and within yfdI, DNase I footprinting assays confirmed that oxidized O xyR bound to the predicted site centered 54 bp upstream of the dsbG gene an d 238 bp upstream of a known OxyR binding site in the promoter region of th e divergently transcribed ahpC gene. Although the new binding site was near dsbG, Northern blotting and primer extension assays showed that OxyR bindi ng to the dsbG-proximal site led to the induction of a second ahpCF transcr ipt, while OxyR binding to the ahpCF-proximal site leads to the induction o f both dsbG and ahpC transcripts. Oxidized OxyR binding to the predicted si te centered 40 bp upstream of the fhuF gene was confirmed by DNase I footpr inting, but these assays further revealed a second higher-affinity site in the fhuF promoter, Interestingly, the two OxyR sites in the fhuF promoter o verlapped with two regions bound by the Fur repressor. Expression analysis revealed that fhuF was repressed by hydrogen peroxide in an OxyR-dependent manner, Finally, DNase I footprinting experiments showed OxyR binding to th e site predicted to be within the coding sequence of yfdI, These results de monstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the E, call genome.