FIG 4.

Assay of PerR cysteine oxidation in 40 μM H₂O₂-treated cells. (A) A 6×His tag was fused to the C terminus of perR (KEGG accession number I872_05555) to construct the S. oligofermentans PerR-6×His strain. Using the same approach described in the legends to Fig. 3A and B, redox Western blotting detected cysteine oxidation in the 6×His-tagged PerR protein using the anti-His tag antibody. U and L at the gel left indicate upper and lower protein bands, respectively. (B) A serine substitution of either Cys139 or Cys142 was constructed on a shuttle plasmid (pDL278-perR-6×His), and the plasmid was transformed into the perR deletion strain to construct the perR::pDL278-perRC139S-6×His (C139S) and perR::pDL278-perRC142S-6×His (C142S) strains. The perR deletion mutant harboring pDL278-perR-6×His (WT) was included as a control. The three strains were anaerobically cultured and then treated with 40 μM H₂O₂. Redox Western blotting, as described in the legend to Fig. 3A, was carried out to detect the oxidation of the PerR mutants. (C) The PerR-6×His strain was anaerobically cultured, and cells were treated with 40 μM H₂O₂ for 1 min and 5 min. Using the methods described in the legend to Fig. 3A, the cysteine oxidation of PerR was detected by redox Western blotting. (D) The 6×His-tagged PerR protein was immunoprecipitated from the statically grown PerR-6×His strain as described in Materials and Methods and then resolved on an 18% nonreducing SDS-PAGE gel. The protein band was then subjected to differential alkylation and LC-MS/MS analysis. (Top) Representative MS/MS spectra of the triply charged peptide ions at m/z 863.7217 and 862.7243, corresponding to reduced (left) and oxidized (right) NDTTTYYDFMGHQHLNVIC¹⁰⁰EK peptide fragments, respectively. (Middle) Representative MS/MS spectra of triply charged peptide ions at m/z 989.1003 and 992.4445, corresponding to both Cys100 and Cys103 reduced (left) and oxidized (right) NDTTTYYDFMGHQHLNVIC¹⁰⁰EKC¹⁰³GR peptide fragments, respectively. (Bottom) The MS/MS spectra represent triple- and quintuple-charged peptide ions at m/z 1038.1281 and 622.0834, respectively, corresponding to both Cys139 and Cys142 reduced (left) and oxidized (right) SQMVVYGIC¹³⁹PEC¹⁴²AQQEQVASHHHHHH peptide fragments. The reduced and oxidized cysteine residues were ¹³C carboxymethylated and ¹²C carbamidomethylated, respectively.