Role of the bacillithiol redox buffer for redox control in Firmicutes bacteria

• Applicant: Professorin Dr. Haike Antelmann
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2012 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 220923428

Prokaryotic and eukaryotic cells are encountered by reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive electrophilic species (RES) which are generated during respiration and metabolism or supplied externally by toxic compounds, such as antibiotics and xenobiotics. Pathogenic bacteria have to cope especially with the strong oxidant hypochloric acid that is released by activated macrophages during the infection process. In a recent study, we have studied changes in the transcriptome and redox proteome caused by NaOCl in Bacillus subtilis. We discovered important roles of the bacillithiol (Cys-GlcN-Malate, BSH) redox buffer in B. subtilis in redox regulation and protection of active site Cys residues of essential enzymes against irreversible oxidations under disulfide stress conditions. NaOCl stress caused S-bacillithiolation of the redox-sensing MarR-type repressor OhrR and of four enzymes of the methionine biosynthesis pathway (MetE, YxjG, PpaC and SerA) that protect cells against NaOCl toxicity. In eukaryotes and Escherichia coli, protein S-glutathionylation has emerged as a major cellular regulatory mechanism and the inactivation of several metabolic enzymes is caused by S-glutathionylation in response to oxidative stress. Thus, we aim in this grant to investigate the global physiological role of BSH in redox regulation of cytoplasmic and regulatory proteins by S-bacillithiolation among BSH-producing Firmicutes bacteria, including industrial important Bacillus species (Bacillus halodurans, Bacillus clausii, Bacillus megaterium, Bacillus amyloliquefaciens,) and Staphylococcus carnosus, the radioresistant Deinococcus radiodurans as well as the pathogenic Bacillus cereus. We will use 2D gel-based redox proteomics methods, shotgun-LC-MS/MS analysis and MS-based stable isotop metabolic labelling with 14N/15N-ammonium coupled to immunoprecipitation of BSH-modified peptides and proteins to identify and quantify the extent of S-bacillithiolations in response to ROS. Our first proteome-wide studies have identified novel bacilliredoxins (YphP, YqiW and YtxJ) as target for S-bacillithiolation that could function as thiol-disulfide oxidoreductases (bacilliredoxins, Brx) in reduction of S-bacillithiolated proteins. Thus, the functions and substrates of novel BSH-related bacilliredoxins will be analyzed using phenotype analyses of brx mutant strains and novel gel-based and gel-free redox proteomics methods.

• DFG Programme: Research Grants