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.

FIG. 1

Sequence of the ahpC and dsbG promoters. The ahpC and dsbG transcription starts are marked by black arrows, and the starts of the corresponding ORFs are denoted by white arrows. The DNase I footprints for OxyR binding to the top and bottom strands are indicated by the dark gray boxes. The DNase I footprint for RNA polymerase binding adjacent to the dsbG-proximal OxyR binding site on the bottom strand is indicated by the light gray box. The locations of the two predicted oxyR sites are shown by sequence walkers (12), in which the rectangles surrounding the T's at positions 637851 and 638089 indicate the centers of the binding sites. A sequence walker consists of a string of letters in which the height of each letter shows the contribution that the corresponding base would make to the average sequence conservation shown by the sequence logo of all binding sites (10). The sequence walker is given on a scale of bits of information. The scale, from −3 bits up to the maximum conservation at +2 bits, is given by the rectangle surrounding the T. Positively contributing bases are above the zero line, and negatively contributing bases are below the line. By using bits, the heights of all letters can be added together to obtain the information content of a site.

FIG. 2

DNase I footprinting assays of oxidized OxyR binding to the top and bottom strands of the dsbG promoter in the absence of RNA polymerase (A) and to the top strand in the presence of RNA polymerase (B). The regions protected by OxyR are indicated by the brackets in panel A. All samples were run in parallel with Maxam-Gilbert G/A sequencing ladders. (A) For OxyR binding to the top strand relative to the dsbG promoter, the 230-bp EcoRI-BamHI fragment of pGSO123 was labeled with ³²P at the EcoRI site. For OxyR binding to the bottom strand relative to the dsbG promoter, the ³²P-labeled primer 710 and unlabeled primer 709 were used to PCR amplify a 430-bp fragment containing both the ahpC and dsbG promoter sequences. (B) For OxyR and RNA polymerase binding to the top strand relative to the dsbG promoter, a 250-bp fragment was PCR amplified from pGSO124 using primers 710 and 726. The amplified fragment was digested with EcoRI, labeled with ³²P, and then digested with SmaI.

FIG. 3

Primer extension assays of ahpC and dsbG expression in wild-type and ΔoxyR mutant strains grown in LB medium. Exponential-phase cultures were split into two aliquots: one aliquot was left untreated, and the other was treated with 1 mM hydrogen peroxide. The cells were harvested after 10 min, total RNA was isolated, and primer extension assays were carried out with primer 709 specific to ahpC and primer 610 specific to dsbG. The neighboring sequencing reactions were carried out with the same primers.

FIG. 4

Sequence of the fhuF promoter. The fhuF transcription start is marked by the black arrow, and the start of the FhuF ORF is denoted by the white arrow. The DNase I footprints for OxyR binding to the top and bottom strands are indicated by the dark gray boxes. The DNase I footprints for Fur binding to the top strands (K. A. Lewis, B. Doan, M. Zheng, G. Storz, and T. D. Schneider, unpublished data) are indicated by the light gray boxes. The locations of the two predicted oxyR sites are shown by sequence walkers (12), in which the rectangles surrounding the A at position 4603357 and the T at position 4603273 indicate the centers of the binding sites. The site with an information content of 11.8 bits is designated fhuF-2, and the site with an information content of 4.8 bits is designated fhuF-1.

FIG. 5

DNase I footprinting assays of oxidized OxyR binding to the top and bottom strands of the fhuF promoter. The regions protected by OxyR on both strands are indicated by the brackets. The 240-bp BamHI-EcoRI fragment of pGSO129 was labeled with ³²P at either the BamHI site (top strand) or the EcoRI site (bottom strand). The samples were run in parallel with Maxam-Gilbert G/A sequencing ladders.

FIG. 6

Primer extension assays of fhuF expression in wild-type, ΔoxyR, and Δfur strains grown in LB medium without (lanes 1 to 6) and with (lanes 7 to 12) 1 mM 2,2′-dipyridyl. Exponential-phase cultures were split into two aliquots: one aliquot was left untreated, and the other was treated with 1 mM hydrogen peroxide. The cells were harvested after 10 min, total RNA was isolated, and primer extension assays were carried out with primer 706 specific to fhuF. The neighboring sequencing reactions were carried out with the same primer.

FIG. 7

Sequence of the yfdI gene. The YfdI ORF, which extends from position 2467151 to 2468482, is indicated by the white arrow. The DNase I footprints for OxyR binding to the top and bottom strands are indicated by the dark gray boxes. The location of the predicted oxyR site is shown by a sequence walker (12), in which the rectangle surrounding the A at position 2467330 indicates the center of the binding site.

FIG. 8

DNase I footprinting assays of oxidized OxyR binding to the top and bottom strands of the yfdI gene. The regions protected by OxyR on both strands are indicated by the brackets. The 180-bp BamHI-EcoRI fragment of pGSO130 was labeled with ³²P at either the BamHI site (top strand) or the EcoRI site (bottom strand). The samples were run in parallel with Maxam-Gilbert G/A sequencing ladders.