Regulation of hydrogen peroxide-dependent gene expression in Rhodobacter sphaeroides: regulatory functions of OxyR

Abstract

Genome-wide transcriptome profiling was used to reveal hydrogen peroxide (H(2)O(2))-dependent regulatory mechanisms in the facultatively photosynthetic bacterium Rhodobacter sphaeroides. In this study we focused on the role of the OxyR protein, a known regulator of the H(2)O(2) response in bacteria. The transcriptome profiles of R. sphaeroides wild-type and oxyR mutant strains that were exposed to 1 mM H(2)O(2) for 7 min or were not exposed to H(2)O(2) were analyzed. Three classes of OxyR-dependent genes were identified based on their expression patterns in the wild type of oxyR mutant strains with differing predicted roles of oxidized and reduced OxyR as activators of transcription. DNA binding studies revealed that OxyR binds upstream of class I genes, which are induced by H(2)O(2) and exhibit similar basal levels of expression in the wild-type and oxyR mutant strains. The effect of OxyR on class II genes, which are also induced by H(2)O(2) but exhibit significantly lower basal levels of expression in the wild-type strain than in the mutant, is indirect. Interestingly, reduced OxyR also activates expression of few genes (class III). The role of reduced OxyR as an activator is shown for the first time. Our data reveal that the OxyR-mediated response is fast and transient. In addition, we found that additional regulatory pathways are involved in the H(2)O(2) response.

FIG. 1.

Binding of oxidized OxyR (OxyR_ox) and reduced OxyR (OxyR_red) to the upstream regions of RSP1408 (A), RSP2638 (B), and RSP3751 (C), as determined by gel mobility shift assays. To generate reduced conditions, 200 mM DTT was added to the binding reaction mixtures. The following amounts of OxyR were added to the reaction mixture: 50 ng (lanes 1 and 4), 100 ng (lanes 2 and 5), and 150 ng (lanes 3 and 6). Lane C contained the control without OxyR.

FIG. 2.

(A) Footprint assay of oxidized OxyR (OxyR_ox) and reduced OxyR (OxyR_red) for the upstream region of the R. sphaeroides class III gene RSP3751. To generate reduced conditions, 200 mM DTT was added to the binding reaction mixtures. The regions protected by OxyR are indicated by vertical lines on the left; the numbers indicate the binding positions with respect to the translational start site. Lane c contained the control without OxyR. Lanes G, A, T, and C show the sequencing reaction. (B) Sequence of the upstream region of RSP3751. The start of the RSP3715 coding sequence is indicated by an arrow. Binding sites of reduced OxyR are underlined.

FIG. 3.

Model describing the OxyR mode of action for class I and class III genes. Based on microarray data and DNA-protein binding studies, we concluded that oxidized OxyR (in the presence of H₂O₂) and reduced OxyR (no-stress conditions) have the following direct effects on the expression of OxyR-dependent genes. For class I genes, OxyR activates gene expression in the presence of H₂O₂. Class I genes are activated by binding of oxidized OxyR to the upstream region, whereas under no-stress conditions reduced OxyR binds to the DNA but has no regulatory effect on the expression of these genes. For class III genes, reduced OxyR binds to the upstream regions and activates class III genes under no-stress conditions. Oxidized OxyR does not bind or only weakly binds to the upstream region of these genes, and gene expression decreases in the presence of H₂O₂.