Regulation of thiol-specific electrophile resistance mechanisms in Bacillus subtilis
Final Report Abstract
Bacillus subtilis and related Firmicutes bacteria encode redox-sensing MarR/OhrR-family and MarR/DUF24-family regulators to sense Reactive oxygen and electrophilic species (ROS and RES). We studied the redox-sensing mechanisms of novel MarR/DUF24-type regulators in B. subtilis. The regulatory mechanisms of HypR, YodB and CatR were characterized that sense and respond to quinones, diamide and hypochlorite stress. HypR is the first DUF24-family regulator whose crystal structure was resolved in collaboration with Prof. Winfried Hinrichs and Dr. Gottfried Palm (University of Greifswald). HypR senses specifically disulfide stress and controls positively expression of the flavin oxidoreductase HypO after NaOCl and diamide stress. HypR resembles a 2-Cys-type regulator with a redox-sensing nucleophilic Cys14 residue and a second C-terminal Cys49. The Cys14 residue is conserved among the DUF24-family, has a lower pK a of 6.36 and is essential for activation of hypO transcription by disulfide stress. HypR is activated by Cys14-Cys49' intersubunit disulfide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR, a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ~4 Å towards each other. B. subtilis encodes further MarR/DUF24-family members including the paralogous YodB and CatR repressors. YodB controls the azoreductase AzoR1, the nitroreductase YodC and the Spx regulator that are up-regulated by quinones and diamide and confer resistance to the electrophiles. YodB resembles a 2-Cys-type MarR/DUF24-family regulator with three Cys residues (Cys6, Cys101 and Cys108). Using a yodBC6S mutant, it was shown that the conserved Cys6 is essential for redox sensing of diamide and MHQ in vivo. YodB senses diamide and quinones by Cys6-Cys101' or Cys6-Cys108' intersubunit disulfides in vivo. YodB and its paralog YvaP (CatR) were further identified as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Only inactivation of both regulators, YodB and CatR results in full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis showed that CatR protects two inverted repeats as operator sites BS1 and BS2 with the consensus sequence TTAC-N5-GTAA overlapping the -35 promoter region and the transcriptional start site. The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue, that is required for redox-sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data revealed that CatR forms intermolecular disulfide in response to diamide and quinones in vitro. In conclusion, HypR, YodB and CatR are controlled by 2-Cys-type thiol-disulfide redox switches to sense disulfide and RES stress and to control RES detoxification enzymes. One main part of this project was the analysis of the regulatory mechanisms and posttranslational thiol-modifications that are caused by the strong oxidant hypochlorite in B. subtilis. Using transcriptomics, redox proteomics and LTQ-Orbitrap mass spectrometry analysis, the changes in the gene expression profile and the targets for reversible thiol-modifications after NaOCl stress were analyzed in B. subtilis. The transcriptome profile after NaOCl stress was indicative of disulfide stress and overlapped strongly with the response to diamide. NaOCl stress caused the induction of the thiol- and oxidative stress-specific Spx, CtsR, PerR and OhrR regulons. Thiol redox proteomics identified only few NaOCl-sensitive proteins with reversible thiol-oxidations that included GapA, MetE, AroA, MtnA and PurQ. Using mass spectrometry, eleven proteins were identified that were oxidized to mixed BSH protein disulfides (S-bacillithiolated) in B. subtilis cells after NaOCl-exposure. These S-bacillithiolated proteins included the OhrR repressor and two methionine synthases MetE and YxjG and we showed that S-bacillithiolation functions as redox switch mechanism in response to NaOCl stress. S-bacillithiolation of the OhrR repressor leads to upregulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE and YxjG causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. Our results showed for the first time an important role of the BSH redox buffer in redox control and protein thiol protection under oxidative stress conditions by S-bacillithiolation of redox-sensing regulators and main metabolic and essential enzymes.
Publications
- 2010. Biosynthesis and functions of Bacillithiol: a major low molecular weight thiol in Bacilli, Proc. Natl. Acad. Sci. 107: 6482-6486
Gaballa, A., Newton, J., Antelmann, H., Parsonage, D., Upton, H., Rawat, M., Claiborne, A., Fahey, R., and Helmann, J.D.
- 2010. The paralogous MarR/DUF24-family repressors YodB and CatR (YvaP) control expression of the catechol dioxygenase CatE in Bacillus subtilis. J. Bacteriol. 192: 4571-4581
Chi, B.K., Kobayashi, K., Albrecht, D., Hecker, M., and Antelmann, H.
- 2010. The redox-sensing regulator YodB senses diamide and quinones via a thiol-disulfide switch in Bacillus subtilis. Proteomics 10: 3155-3164
Chi, B.K., Albrecht, D., Gronau, K., Becher, D. Hecker, M., and Antelmann, H.
- 2011. S- bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics. Mol Cell Proteomics 10: M111.009506
Chi, B.K., Gronau, K., Mäder, U., Hessling, B., Becher, D., and Antelmann, H.
- 2011. Thiol-based redox switches and gene regulation. Forum Review Article on “Redox switches”. Antioxidants & Redox signaling 14: 1049-63
Antelmann, H., and Helmann, J.D.
- 2012. Structural insights into the redox-switch mechanism of the MarR/DUF24-family regulator HypR. Nucleic Acid Research 40: 4178-92
Palm, G., Chi, B.K., Waack, P., Gronau, K., Becher, D., Albrecht, D., Hinrichs, W., Read, R.J. and Antelmann, H.
(See online at https://doi.org/10.1093/nar/gkr1316)
