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

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.

SUBMITTER: Gu M

PROVIDER: S-EPMC3071027 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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

Gu Mianzhi M Imlay James A JA

Molecular microbiology 20110112 5

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 ...[more]

PMID: 21226770

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Project description:The soxR locus of Escherichia coli K12 mediates transcriptional activation of a complex oxidative stress regulon in response to superoxide-generating (redox-cycling) agents. We have cloned the soxR locus, which is positioned near the uvrA gene at 92.2 min on the genetic map, by monitoring complementation of a delta soxR mutation. Subclones from the soxR region in the delta soxR strain simultaneously restored cellular resistance to the redox-cycling agent phenazine methosulfate and inducibility of at least two of the regulon proteins, glucose-6-phosphate dehydrogenase and endonuclease IV, by paraquat, another redox-cycling agent. DNA sequence analysis revealed the presence of two genes involved in activating the soxR regulon. These genes, named soxR and soxS, are arranged divergently with their 5' ends separated by only 85 bp. The predicted 12.9-kDa SoxS protein is related to the AraC family of one-component gene regulators, but corresponds only to the putative DNA-binding regions of these proteins. The 17.1-kDa SoxR protein bears significant homology only to the MerR family of proteins including a predicted DNA-binding helix-turn-helix and a cluster of cysteine residues positioned similarly to those that regulate the activity of MerR in response to Hg2+. This suggests that SoxR could be a metal-binding gene regulator that acts as the intracellular sensor for superoxide. SoxS is evidently the proximal activator of the regulon genes: antibiotic resistance and high-level expression of at least three of the regulon proteins was effected in vivo by the individual expression of SoxS, but not of SoxR, whether or not the cells were exposed to paraquat. These data, together with the recently reported paraquat-inducibility of the soxS gene (Wu, I., and Weiss, B. (1990) J. Bacteriol. 173, 2864-2871), indicate that SoxR and SoxS may constitute a novel type of two-component regulatory system in which the two proteins act sequentially to activate transcription of the various regulon genes in response to superoxide stress.

Project description:In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461-467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe(3+/2+)reduction potentials that vary by approximately 1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe(3+/2+) reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.

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

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

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