Phase variation of Ag43 is independent of the oxidation state of OxyR

Abstract

OxyR is a DNA binding protein that differentially regulates a cell's response to hydrogen peroxide-mediated oxidative stress. We previously reported that the reduced form of OxyR is sufficient for repression of transcription of agn43 from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of agn43. Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses agn43 transcription. In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of agn43. OxyR(ox) repressed in vitro transcription but only from an unmethylated agn43 template. The -10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox). Furthermore, OxyR(ox) bound to the agn43 regulatory region DNA with an affinity similar to that for the regulatory regions of katG and oxyS, which are activated by OxyR(ox), indicating that binding at agn43 can occur at biologically relevant concentrations. OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at agn43 is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR.

FIG. 1.

Different binding properties of wild-type and mutant OxyR to unmethylated agn43 regulatory region. Results obtained from EMSA with cell extracts containing OxyR are shown in panel A (lanes 2 to 5), with OxyR(H198R) in panel B, and with OxyR(A233V) in panel C. Amounts of total protein added were 0 μg (lanes 1 and 6), 0.42 μg (lanes 2), 0.85 μg (lanes 3), 2.55 μg (lanes 4), and 4.25 μg (lanes 5). Also shown is the shift obtained with 7 ng of purified, oxidized OxyR (A, lane 7).

FIG. 2.

Oxidized OxyR represses transcription from an unmethylated agn43 template in vitro. Template DNA was either unmethylated (lanes 1 to 3) or methylated (lanes 4 to 6). OxyR(ox) was added as indicated before RNA polymerase to a final concentration of 9.5 nM (lanes 2 and 5) or 19 nM (lanes 1 and 6). No OxyR was added to reactions shown in lanes 3 and 4. “Control” refers to a transcript obtained from a vector-derived promoter. The size of the agn43 transcription product is 220 nt. Lane 2 was obtained from a different exposure of the same gel.

FIG. 3.

The DNase I footprint of oxidized OxyR is similar to that of OxyR(C199S) at agn43 DNA. Footprints were obtained using a final concentration of 12 nM OxyR(ox) (lanes 1 and 4) or 10 nM OxyR(C199S) (lanes 3 and 6). Template DNA was unmethylated agn43 DNA (lanes 1 to 3) and oxyRS DNA (lanes 4 to 6). For the agn43 sequence, the position of the three GATC sequences is shown. The direction of transcription of oxyR and oxyS is shown for the oxyRS footprint, as well as the position of the OxyR binding motifs O1 through O5, as designated previously by Toledano et al. (34).

FIG. 4.

The affinity of oxidized OxyR for unmethylated agn43 DNA is similar to that for katG and oxyRS DNA. The percent shifted DNA in an EMSA with increasing amounts of purified wild-type OxyR is shown. Eight nanograms of OxyR corresponds to a final concentration of 12 nM. Template DNA consisted of oxyRS (open diamonds), katG (open squares), and unmethylated agn43 regulatory regions (closed triangles). Data from two independent series of experiments are shown.

FIG. 5.

Schematic model for regulation of agn43 transcription. Vertical lines indicate the GATC sequences, and methylation is indicated by CH₃. The oxidized and the reduced forms of OxyR are as indicated, and the transcription start site is indicated by an arrow. The ON phase (A) is obtained if the three GATC sequences in regulatory region are methylated. The OFF phase is obtained by OxyR-dependent repression. Both the oxidized (C) and reduced forms (B) of OxyR repress transcription from the unmethylated agn43 template. Neither the oxidized nor reduced form of OxyR could bind to the methylated agn43 template.