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

Abstract

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.

Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

“…The fiu::lacZ fusion requires seven times more ferrozine to yield half of the derepression compared with fhuF::lacZ (32). Consistently, four overlapping Fur boxes were found in the promoter region of the fiu operon (39), and only two were found in the fhuF promoter (40). In our normal growing conditions, Fur was active.…”

Regulation of inducible peroxide stress responses

“…In this case, activated OxyR mediates repression of this operon in response to oxidative stress. Remarkably, in an oxyR mutant, this operon is induced by high concentrations of H 2 O 2 (1 mM), perhaps as a result of inactivation of bound Fur protein (Zheng et al, 2001a). In summary, the Fur-and PerR-like regulators may form a continuum of regulators that vary from peroxide-sensing repressors that require a metal ion for DNA binding to metal-sensing repressors that may also display some sensitivity to ROS.…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…Interestingly, expression of the fur gene is induced by both the OxyR and SoxS proteins (129). Positive control by OxyR is evidently required because H 2 O 2 tends to inactivate the ferrous-Fur complex (72), perhaps by oxidation of the ferrous iron cofactor.…”