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Journal of Bacteriology, August 2001, p. 4571-4579, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4571-4579.2001

Computation-Directed Identification of OxyR DNA Binding Sites in Escherichia coli

Ming Zheng,1,dagger Xunde Wang,1 Bernard Doan,1 Karen A. Lewis,2 Thomas D. Schneider,2 and Gisela Storz1,*

Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892,1 and Laboratory of Experimental and Computational Biology, National Cancer Institute, Frederick, Maryland 217022

Received 15 March 2001/Accepted 15 May 2001

A computational search was carried out to identify additional targets for the Escherichia coli OxyR transcription factor. This approach predicted OxyR binding sites upstream of dsbG, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of fhuF, encoding a protein required for iron uptake; and within yfdI. DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the dsbG gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed ahpC gene. Although the new binding site was near dsbG, Northern blotting and primer extension assays showed that OxyR binding to the dsbG-proximal site led to the induction of a second ahpCF transcript, while OxyR binding to the ahpCF-proximal site leads to the induction of both dsbG and ahpC transcripts. Oxidized OxyR binding to the predicted site centered 40 bp upstream of the fhuF gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in the fhuF promoter. Interestingly, the two OxyR sites in the fhuF promoter overlapped 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 the site predicted to be within the coding sequence of yfdI. These results demonstrate 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. coli genome.

* Corresponding author. Mailing address: NIH, Building 18T, Room 101, 18 Library Dr., MSC 5430, Bethesda, MD 20892-5430. Phone: (301) 402-0968. Fax: (301) 402-0078. E-mail: storz{at}helix.nih.gov.

dagger Present address: Biochemical Science and Engineering, Central Research and Development, E. I. DuPont de Nemours and Company, Wilmington, DE 19880-0328.

Journal of Bacteriology, August 2001, p. 4571-4579, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4571-4579.2001


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