Regulation of thiol-specific electrophile resistance mechanisms in Bacillus subtilis

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The novel MarR-type repressors YodB, YvaP and MhqR confer resistance to electrophilic quinones and diamide in Bacillus subtilis via the control of paralogous thiol-dependent dioxygenases and oxidoreductases. YodB and YvaP are redox-sensitive MarR/DUF24 family regulators that sense quinone-like electrophiles and diamide via the conserved N-terminal Cys residue. Previous data suggest that the YodB repressor is regulated via thiol-(S)-alkylation in response to quinones in vitro. Proteomic and thiol-redox proteomic studies revealed that quinones irreversibly alkylate and aggregate thiol-containing proteins in vivo. In contrast, diamide treatment leads to reversible disulfide bond formation in vivo. In this project, the detailed mechanisms of quinone and diamide sensing by YodB and YvaP will be investigated in vitro and in vivo. In addition, the regulatory mechanisms that are involved in derepression of MhqR are further focused in this project. The functions of the novel thiol-dependent dioxygenases and reductases in quinone and diamide detoxification will be analyzed. Besides YodB and YvaP, six other YodB paralogs (YdeP, YybR, YkvN, YdzF, YcdF and HxlR) are encoded in the genome of B. subtilis which will be characterized using genome-wide analyses and detailed biochemical and genetic approaches. Besides quinones, carbonyl compounds (e.g. formaldehyde or methylglyoxal) are natural electrophiles, that most likely modify thiol-containing proteins via the thiol-(S)-alkylation chemistry, depleting the cellular thiol pool. The novel MerR/NmlR-family regulator AdhR (YraB) was identified as aldehyde-specific sensor and activation of AdhR required the conserved Cys52 in vivo. AdhR controls a thiol-dependent formaldehyde dehydrogenase (AdhA) and the cysteine proteinase YraA, both of which protect cells against FA toxicity. The identification of post-translational modifications caused by formaldehyde and methylglyoxal in AdhR and cellular protein thiolates is another focus of this project. The functions of AdhA and YraA in detoxification of aldehydes and repair or degradation of damaged thiol-containing proteins will be investigated.