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. 2011 Mar;79(5):1136-50.
doi: 10.1111/j.1365-2958.2010.07520.x. Epub 2011 Jan 12.

The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide

Mianzhi Gu  1 , James A Imlay
Affiliations

The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide

Mianzhi Gu et al. Mol Microbiol. 2011 Mar.

Abstract

When Escherichia coli is exposed to redox-cycling drugs, its SoxR transcription factor is activated by oxidation of its [2Fe-2S] cluster. In aerobic cells these drugs generate superoxide, and because superoxide dismutase (SOD) is a member of the SoxRS regulon, superoxide was initially thought to be the activator of SoxR. Its many-gene regulon was therefore believed to comprise a defence against superoxide stress. However, we found that abundant superoxide did not effectively activate SoxR in an SOD⁻ mutant, that overproduced SOD could not suppress activation by redox-cycling drugs, and that redox-cycling drugs were able to activate SoxR in anaerobic cells as long as alternative respiratory acceptors were provided. Thus superoxide is not the signal that SoxR senses. Indeed, redox-cycling drugs directly oxidized the cluster of purified SoxR in vitro, while superoxide did not. Redox-cycling drugs are excreted by both bacteria and plants. Their toxicity does not require superoxide, as they poisoned E. coli under anaerobic conditions, in part by oxidizing dehydratase iron-sulfur clusters. Under these conditions SoxRS induction was protective. Thus it is physiologically appropriate that the SoxR protein directly senses redox-cycling drugs rather than superoxide.

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Figures

Fig. 1
Fig. 1. Structures of redox-cycling drugs
Left, paraquat (PQ, methyl viologen); middle, menadione (MD); right, phenazine methosulfate (PMS).
Fig. 2
Fig. 2. The SoxRS response is not activated by superoxide
(A) Strains AB1157 (wild type) (●), PN134 (sodA sodB) (■) and RB2 (sodA sodB soxS) (▲) were pre-cultured in anaerobic LB medium to log phase. At time zero, the pre-cultures were diluted into aerobic medium, and growth was monitored. (B) Cultures of SOD-proficient strain (SOD+) TN530 (soxS::lacZ) and SOD-deficient strain (SOD−) AS358 (sodA sodB soxS::lacZ) were grown in LB medium to an OD600 of 0.1. The cultures were each split into two flasks, 160 μM PQ was added to one, and the other was left untreated. All cultures were aerated for an additional 45 min. The error bars represent the range of activity measured for three independent cultures. (C) Strains AS395 (sodA soxS::lacZ) (closed symbols) and AS396 (sodA soxS::lacZ psodB) (open symbols) were grown aerobically in LB medium to an OD600 of 0.05. The cultures were diluted into flasks containing PMS (diamonds), MD (triangles) or PQ (squares). The cultures were further incubated at 37°C for 60 min. Solution assays showed that SOD activity was twenty-fold higher in strains carrying the psodB plasmid.
Fig. 2
Fig. 2. The SoxRS response is not activated by superoxide
(A) Strains AB1157 (wild type) (●), PN134 (sodA sodB) (■) and RB2 (sodA sodB soxS) (▲) were pre-cultured in anaerobic LB medium to log phase. At time zero, the pre-cultures were diluted into aerobic medium, and growth was monitored. (B) Cultures of SOD-proficient strain (SOD+) TN530 (soxS::lacZ) and SOD-deficient strain (SOD−) AS358 (sodA sodB soxS::lacZ) were grown in LB medium to an OD600 of 0.1. The cultures were each split into two flasks, 160 μM PQ was added to one, and the other was left untreated. All cultures were aerated for an additional 45 min. The error bars represent the range of activity measured for three independent cultures. (C) Strains AS395 (sodA soxS::lacZ) (closed symbols) and AS396 (sodA soxS::lacZ psodB) (open symbols) were grown aerobically in LB medium to an OD600 of 0.05. The cultures were diluted into flasks containing PMS (diamonds), MD (triangles) or PQ (squares). The cultures were further incubated at 37°C for 60 min. Solution assays showed that SOD activity was twenty-fold higher in strains carrying the psodB plasmid.
Fig. 2
Fig. 2. The SoxRS response is not activated by superoxide
(A) Strains AB1157 (wild type) (●), PN134 (sodA sodB) (■) and RB2 (sodA sodB soxS) (▲) were pre-cultured in anaerobic LB medium to log phase. At time zero, the pre-cultures were diluted into aerobic medium, and growth was monitored. (B) Cultures of SOD-proficient strain (SOD+) TN530 (soxS::lacZ) and SOD-deficient strain (SOD−) AS358 (sodA sodB soxS::lacZ) were grown in LB medium to an OD600 of 0.1. The cultures were each split into two flasks, 160 μM PQ was added to one, and the other was left untreated. All cultures were aerated for an additional 45 min. The error bars represent the range of activity measured for three independent cultures. (C) Strains AS395 (sodA soxS::lacZ) (closed symbols) and AS396 (sodA soxS::lacZ psodB) (open symbols) were grown aerobically in LB medium to an OD600 of 0.05. The cultures were diluted into flasks containing PMS (diamonds), MD (triangles) or PQ (squares). The cultures were further incubated at 37°C for 60 min. Solution assays showed that SOD activity was twenty-fold higher in strains carrying the psodB plasmid.
Fig. 3
Fig. 3. The SoxRS response protects a [4Fe–4S] dehydratase by inducing yggX
Anaerobic cultures of SOD-deficient strains growing in LB plus 0.2% gluconate and 100 μg/ml ampicillin were diluted into the same aerobic medium to an OD600 of 0.05. IPTG was added (100 μM). The cultures were grown at 37 °C for ~3 hours to an OD600 of 0.4, and the activity of 6-phosphogluconate dehydratase (Edd) was determined. GS09, SOD− + vector ; GS11, SOD− psoxS; GS10, SOD− pyggX; GS07, SOD− yggX psoxS. Edd activity in a wild type strain was defined as 100%.
Fig. 4
Fig. 4. SoxRS is activated by redox-cycling drugs under anaerobic conditions if terminal oxidants are present
(A) Strain TN530 (soxS::lacZ) was grown aerobically in LB medium (◆); anaerobically in LB medium (■), in LB/nitrate medium (▲), or in LB/fumarate medium (●). Strain GS69 (narG soxS::lacZ) was grown anaerobically in LB/nitrate medium (△); strain GS70 (frd soxS::lacZ) was grown anaerobically in LB medium (□) or LB/fumarate medium (○). At an OD600 of 0.07, PQ was added, and the cultures were further incubated at 37°C for one hour. (B) Strain TN530 (soxS::lacZ) was grown anaerobically in LB medium (■), or in LB/fumarate medium (●). Strain GS67 (nuo ndh soxS::lacZ) (◇) and GS68 (ubi men soxS::lacZ) (○) were grown anaerobically in LB/fumarate medium.
Fig. 4
Fig. 4. SoxRS is activated by redox-cycling drugs under anaerobic conditions if terminal oxidants are present
(A) Strain TN530 (soxS::lacZ) was grown aerobically in LB medium (◆); anaerobically in LB medium (■), in LB/nitrate medium (▲), or in LB/fumarate medium (●). Strain GS69 (narG soxS::lacZ) was grown anaerobically in LB/nitrate medium (△); strain GS70 (frd soxS::lacZ) was grown anaerobically in LB medium (□) or LB/fumarate medium (○). At an OD600 of 0.07, PQ was added, and the cultures were further incubated at 37°C for one hour. (B) Strain TN530 (soxS::lacZ) was grown anaerobically in LB medium (■), or in LB/fumarate medium (●). Strain GS67 (nuo ndh soxS::lacZ) (◇) and GS68 (ubi men soxS::lacZ) (○) were grown anaerobically in LB/fumarate medium.
Fig. 5
Fig. 5. By serving as an artificial electron sink, ferricyanide enables anaerobic PMS to activate SoxRS
Strain TN530 (soxS::lacZ) was grown anaerobically in LB medium to an OD600 of 0.05. Where indicated, PMS was added to a final concentration of 25 μM, and potassium ferricyanide was added to final concentrations of 150 μM or 800 μM. All cultures were further incubated anaerobically at 37°C for one hour.
Fig. 6
Fig. 6. The [2Fe–2S] cluster of SoxR is directly oxidized by redox-cycling drugs in vivo and in vitro
(A) Strain XA90/pKOXR containing the soxR expression plasmid was grown in anaerobic LB medium to an OD600 of 0.15. IPTG was added, and the cultures were incubated anaerobically at 37°C for 1.5 hr. Where indicated, PQ was then added, and cultures were further incubated anaerobically at 37°C for 30 min. EPR samples from these intact cells were prepared as described in Materials and Methods. The peaks indicated by arrows represent the EPR signal contributed by reduced SoxR; they are absent from vector control strains (not shown). (B) Purified SoxR protein (15 μM) was reduced with dithionite anaerobically. Excess dithionite was removed (see Materials and Methods). Reduced SoxR was then treated with 25 μM PMS in anerobic buffer at 25°C for 2 min. For EPR sample preparation, see Materials and Methods.
Fig. 6
Fig. 6. The [2Fe–2S] cluster of SoxR is directly oxidized by redox-cycling drugs in vivo and in vitro
(A) Strain XA90/pKOXR containing the soxR expression plasmid was grown in anaerobic LB medium to an OD600 of 0.15. IPTG was added, and the cultures were incubated anaerobically at 37°C for 1.5 hr. Where indicated, PQ was then added, and cultures were further incubated anaerobically at 37°C for 30 min. EPR samples from these intact cells were prepared as described in Materials and Methods. The peaks indicated by arrows represent the EPR signal contributed by reduced SoxR; they are absent from vector control strains (not shown). (B) Purified SoxR protein (15 μM) was reduced with dithionite anaerobically. Excess dithionite was removed (see Materials and Methods). Reduced SoxR was then treated with 25 μM PMS in anerobic buffer at 25°C for 2 min. For EPR sample preparation, see Materials and Methods.
Fig. 7
Fig. 7. PMS blocks the synthesis of branched-chain amino acids in anaerobic cells, and the SoxRS response is protective
Wild type strain (GC4468, panel A) and soxRS mutant (DJ901, panel B) were cultured to early log phase in anaerobic glucose/nitrate medium supplemented with aromatic and sulfur-containing amino acids. Where indicated, branched-chain amino acids (ILV) were included. At time zero, PMS was added and growth was monitored.
Fig. 7
Fig. 7. PMS blocks the synthesis of branched-chain amino acids in anaerobic cells, and the SoxRS response is protective
Wild type strain (GC4468, panel A) and soxRS mutant (DJ901, panel B) were cultured to early log phase in anaerobic glucose/nitrate medium supplemented with aromatic and sulfur-containing amino acids. Where indicated, branched-chain amino acids (ILV) were included. At time zero, PMS was added and growth was monitored.
Fig. 8
Fig. 8. Redox-cycling drugs directly inactivate [4Fe–4S] fumarases
(A) Redox drugs inactivate fumarase B anaerobically in vivo. Strain SJ33 (fumCA) was grown anaerobically in LB medium. When OD600 reached 0.2, 150 μg/ml chloramphenicol was added to stop protein synthesis. Where indicated, 200 μM PQ, 300 μM MD or 30 μM PMS was added. All cultures were further incubated at 37 °C anaerobically for 45 min. (B) PMS directly inactivates purified fumarase A in vitro. Purified fumarase A was incubated anaerobically on ice by itself (●) or with 0.5 μM (▲), 1 μM (◆) or 5 μM (■) PMS. At time points, damage was terminated by the addition of 20 mM L-malate, and fumarase A activities were measured.
Fig. 8
Fig. 8. Redox-cycling drugs directly inactivate [4Fe–4S] fumarases
(A) Redox drugs inactivate fumarase B anaerobically in vivo. Strain SJ33 (fumCA) was grown anaerobically in LB medium. When OD600 reached 0.2, 150 μg/ml chloramphenicol was added to stop protein synthesis. Where indicated, 200 μM PQ, 300 μM MD or 30 μM PMS was added. All cultures were further incubated at 37 °C anaerobically for 45 min. (B) PMS directly inactivates purified fumarase A in vitro. Purified fumarase A was incubated anaerobically on ice by itself (●) or with 0.5 μM (▲), 1 μM (◆) or 5 μM (■) PMS. At time points, damage was terminated by the addition of 20 mM L-malate, and fumarase A activities were measured.
Fig. 9
Fig. 9. Model of SoxRS activation by redox-cycling drugs
Redox-cycling drugs directly activate SoxR by oxidizing its [2Fe–2S] cluster. The oxidized SoxR activates transcription of soxS, which then stimulates expression of the genes in the SoxRS regulon. Continuous activation requires reoxidation of the reduced drug by the oxidized quinone pool of an active respiratory chain.
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