Oxidants induce Escherichia coli MarR glutathionylation in the presence of glutathione

The results of protein thiols reacting with oxidants may be different in the presence or absence of glutathione (GSH). Upon exposure to oxidants, such as Cu²⁺ and polysulfide, the multiple drug resistant regulator MarR dimer in Escherichia coli is believed to form tetramers linked by disulfide bonds between its Cys⁸⁰ thiols. We confirmed this observation in the absence of GSH; however, the MarR-Cys⁸⁰ thiol was primarily glutathionylated in the presence of GSH after MarR was treated with various oxidants, including octasulfur (S₈), Cu²⁺, H₂O₂, ClO^-, and a NO donor. When using S₈ as the oxidizing agent, we identified four pathways to induce MarR-Cys⁸⁰ glutathionylation. Since E. coli contains high concentrations of GSH, MarR is likely glutathionylated instead of forming tetramers inside the cells. When E. coli was exposed to S₈, cellular levels of protein glutathionylation were increased and most MarR was glutathionylated, as shown by Western blot and LC-MS analyses. The glutathionylated MarR displayed reduced affinity to its cognate operator, resulting in the expression of its repressed genes. The results highlight the need to consider the wide presence of GSH when investigating protein thiol modification.

Escherichia coli; Glutathione; MarR; Oxidative stress; Redox regulation; S-Glutathionylation; Supersulfide.