DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide

Abstract

The genome-wide transcription profile of Escherichia coli cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169 E. coli open reading frames. By measuring gene expression in isogenic wild-type and oxyR deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the DeltaoxyR mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in E. coli. For example, the isc operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.

FIG. 1

OxyR-dependent induction of hemH. (A) Sequence of the hemH promoter. The hemH transcription start is marked by the black arrow, and the start of the corresponding ORF is denoted by the white arrow. The DNase I footprints for OxyR binding are indicated by the dark boxes. (B) Primer extension assays of hemH expression in wild-type (MC4100) and ΔoxyR (GSO47) 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 819 specific to hemH. The neighboring sequencing reactions were carried out with the same primer. (C) DNase I footprinting assays of oxidized OxyR binding to the top and bottom strands relative to the hemH promoter. The regions protected by OxyR on both strands are indicated by the brackets. The plasmids carrying the hemH promoter fragment in both orientations were digested with NotI, labeled with ³²P, and then digested with BamHI to give the labeled top and bottom strands. The samples were run in parallel with Maxam-Gilbert G/A sequencing ladders.

FIG. 2

OxyR-dependent induction of the suf operon. (A) Structure of the suf operon and sequence of the sufA promoter. The induction ratios observed for the wild-type and ΔoxyR mutant strains in the microarray experiment are given below each gene. The sufA transcription start is marked by the black arrow, and the start of the corresponding ORF is denoted by a white arrow. The DNase I footprints for OxyR binding are indicated by the dark boxes. Our computational search (41) predicted an OxyR binding site of 9.6 bits centered at position 922026. (B) Primer extension assays of sufA expression in wild-type (MC4100) and ΔoxyR (GSO47) 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 820 specific to sufA. The neighboring sequencing reactions were carried out with the same primer. (C) DNase I footprinting assays of oxidized OxyR binding to the top and bottom strands relative to the sufA promoter. The regions protected by OxyR on both strands are indicated by the brackets. For OxyR binding to the top strand, the ³²P-labeled primer 828 and unlabeled primer 825 were used to PCR amplify a 187-bp fragment. For OxyR binding to the bottom strand, the 378-bp NotI-BamHI fragment of pGSO133 was labeled with ³²P at the BamHI site. The samples were run in parallel with Maxam-Gilbert G/A sequencing ladders.

FIG. 3

Possible OxyR-dependent repression of gntP and uxuAB; b2653, ygaQ, ygaR, and b2656; and ybjC, nfsA/mdaA, and rimK. The gene organization of the corresponding operons is shown. Predicted OxyR binding sites (41) of 7.0 bits centered at position 4549044, 8.8 bits centered at position 2784053, and 5.4 bits centered at position 2784276 are underlined. The confirmed OxyR binding site upstream of the grxA promoter (39) is indicated by the black boxes. The transcription starts documented for grxA and predicted for ybjC are denoted by solid and dotted arrows, respectively. The induction ratios observed for the wild-type and ΔoxyR mutant strains in the microarray experiment are given below each gene.

FIG. 4

oxyS and soxS induction by hydrogen peroxide and paraquat. The figure shows the results of primer extension assays of oxyS and soxS transcript levels in wild-type (MC4100) cells grown in LB medium. An exponential-phase culture was split into aliquots: one aliquot was left untreated, and the other aliquots were exposed to the indicated concentrations of hydrogen peroxide and paraquat. The cells were harvested after 5 min, total RNA was isolated, and primer extension assays were carried out with primer 188 specific to oxyS and primer 823 specific to soxS.

FIG. 5

OxyR- and SoxRS-independent induction of the isc operon. (A) Structure of the isc operon and sequence of the yfhP promoter. The induction ratios observed for the wild-type and ΔoxyR mutant strains in the microarray experiment are given below each gene. The yfhP transcription start is marked by the black arrow, and the start of the corresponding ORF is denoted by a white arrow. (B) Primer extension assays of yfhP expression in wild-type (MC4100), ΔoxyR (GSO47), ΔsoxRS (GSO71), and Δfur (GSO72) 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 686 specific to yfhP. The neighboring sequencing reactions were carried out with the same primer.