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. 1997 Aug 5;94(16):8445-9.
doi: 10.1073/pnas.94.16.8445.

In vivo kinetics of a redox-regulated transcriptional switch

H Ding  1 , B Demple
Affiliations

In vivo kinetics of a redox-regulated transcriptional switch

H Ding et al. Proc Natl Acad Sci U S A. .

Abstract

SoxR is a transcription activator governing a cellular response to superoxide and nitric oxide in Escherichia coli. SoxR protein is a homodimer, and each monomer has a redox-active [2Fe-2S] cluster. Oxidation and reduction of the [2Fe-2S] clusters can reversibly activate and inactivate SoxR transcriptional activity. Here, we use electron paramagnetic resonance spectroscopy to follow the redox-switching process of SoxR protein in vivo. SoxR [2Fe-2S] clusters were in the fully reduced state during normal aerobic growth, but were completely oxidized after only 2-min aerobic exposure of the cells to superoxide-generating agents such as paraquat. The oxidized SoxR [2Fe-2S] clusters were rapidly re-reduced in vivo once the oxidative stress was removed. The in vivo kinetics of SoxR [2Fe-2S] cluster oxidation and reduction exactly paralleled the increase and decrease of transcription of soxS, the target gene for SoxR. The kinetic analysis also revealed that an oxidative stress-linked decrease in soxS mRNA stability contributes to the rapid attainment of a new steady state after SoxR activation. Such a redox stress-related change in soxS mRNA stability may represent a new level of biological control.

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Figures

Figure 1
Figure 1
SoxR [2Fe–2S] clusters are fully reduced in vivo. (A) EPR spectra of the SoxR [2Fe–2S] clusters. Top trace, 10 μM purified [2Fe–2S] SoxR in 500 mM NaCl and 50 mM Hepes (pH 7.6), reduced with freshly prepared 1 mM sodium dithionite. Middle trace, concentrated SoxR-overproducing cells (strain XA90 containing pKOXR). Bottom trace, concentrated cells containing expression vector only (pKEN2). The printing scales of the cell samples were 2-fold less sensitive than that for purified SoxR. (B) Microwave power saturation of the gy band of SoxR [2Fe–2S] clusters in purified SoxR and in vivo.
Figure 2
Figure 2
Oxidation and reduction of SoxR [2Fe–2S] clusters in vivo. (Spectra a and b) SoxR-overproducing cells (XA90 with pKOXR) without or with 100 μM PQ treatment, respectively. (Spectra c and d) Control cells (XA90 with pKEN2) without or with 100 μM PQ treatment, respectively. Cultures treated with or without PQ were aerated at 275 rpm for 20 min before transfer to EPR tubes and freezing. Ten scans were averaged for each spectrum shown. Trace e is the difference spectrum for trace a with trace c subtracted. From the gy band amplitude in trace e, the sample contained ≈0.35 μM reduced SoxR [2Fe–2S] clusters. Trace f is the difference spectrum for trace b with trace d subtracted.
Figure 3
Figure 3
Kinetics of SoxR activation and inactivation in vivo. (A) Kinetics of SoxR [2Fe–2S] oxidation by redox-cycling agents and subsequent re-reduction. SoxR-overproducing cells (XA90 with pKOXR) growing under vigorous aeration were exposed at the indicated time to one of three structurally unrelated compounds: PQ (100 μM), phenazine methosulfate (PMS, 2 μM) and menadione (MD, 100 μM). Aeration was stopped after 10 min as indicated in the figure. Reduced SoxR [2Fe–2S] clusters in vivo were quantified using the amplitude of the gy band in EPR spectra collected as for traces e and f in Fig. 2. Con, cells not treated with redox-cycling agents. (B) Levels of soxS mRNA during SoxR activation and shut off. XA90 cells (containing pKOXR) were exposed to 100 μM PQ as in A, and samples were collected to determine soxS mRNA by Northern blot analysis. (Upper) Ethidium staining of the 1% agarose gel to demonstrate constant loading of total RNA. (Lower) Northern blot of soxS mRNA. (C) Correlation of the redox state of SoxR [2Fe–2S] clusters and soxS mRNA levels. Aliquots of cell culture for EPR measurement at each time point were taken simultaneously with these removed for the soxS mRNA determination shown in B. The amount of soxS mRNA was quantified as described in Materials and Methods.
Figure 4
Figure 4
Half-life of soxS mRNA shortened by oxidative stress. (A) Decay of induced soxS mRNA. Wild-type E. coli (strain GC4468) was used for this experiment. Cells were treated with 100 μM PQ under vigorous aeration (275 rpm) for 20 min before a final concentration of 150 μg/ml rifampicin (from a 300 mg/ml stock dissolved in dimethyl sulfoxide) was added to block RNA synthesis. One half of the culture was then subjected to continuing aeration, and aeration stopped for the other half. (B) Quantitation of soxS mRNA decay. The curves shown were fitted for exponential decays with a half-life of 1.2 min for the aerated samples and 3.2 min for the nonaerated samples.
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