An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein.

Abstract

The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.‐) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe‐SoxR contained non‐heme iron (up to 1.6 atoms per monomer); apo‐SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe‐SoxR indicated that it contains a redox active iron‐sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe‐SoxR and apo‐SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe‐SoxR stimulated transcription initiation at soxS in vitro > 100‐fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe‐SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress.

Functional Analysis of SoxR Residues Affecting Transduction of Oxidative Stress Signals into Gene Expression

“…The [2Fe-2S] centers in purified WT SoxR protein produce a visible absorption spectrum with four maxima at 332, 414, 462, and 548 nm (35,36). Absorption spectra of the purified mutant proteins (data not shown) corroborated the results obtained from in vivo EPR spectroscopy (Fig.…”

Regulation of the soxRS Oxidative Stress Regulon

“…The first evidence in this regard was that the SoxR apoprotein was transcriptionally inactive in vitro (9). Subsequently, it was found that whereas SoxR ox is active in vitro (6,10), it can be inactivated by anaerobic reduction with dithionite and then rapidly restored by autooxidation (3).…”

“…This disassembly may be facilitated in vitro by thiols (6,7). The apoprotein is inactive although it can still bind to DNA (9), whereas the holoprotein can activate the transcription of soxS (9,10). This model was probably promoted because of early failures to verify the alternative model (6).…”

Dual regulation of inaA by the multiple antibiotic resistance (mar) and superoxide (soxRS) stress response systems of Escherichia coli

“…SoxR appears to be a sensor of superoxides and resembles the MerR family of gene regulators (1,38). Recently, an Fe-SoxR protein which activates the transcription of soxS in vitro has been identified (17). The considerable amino acid sequence homologies predicted for the MarA and SoxS proteins from their nucleotide sequences suggest that they may have functions similar to those of AraC-type transcriptional activators (1,6,10).…”