FIG 1.

Requirement for CgMsrA in resistance to multiple oxidative stresses. (A) Survival rates of C. glutamicum strains after being challenged with H₂O₂ (100 mM), CHP (11 mM), CDNB (70 mM), and CdCl₂ (300 μM) for 30 min. (B) The ROS levels in the indicated C. glutamicum strains were measured by using the DCFH-DA fluorescence determination assay after exposure to oxidative reagents. A.U., arbitrary units. (C) Protein carbonyl contents were analyzed by Western blotting with anti-DNP antibody after exposure to various oxidative reagents for 30 min at 30°C. A parallel run stained with Coomassie brilliant blue is shown in the lower gel. Total proteins were extracted from C. glutamicum wild-type cells and msrA mutant cells. (D) β-Galactosidase analyses of msrA promoter activities using the transcriptional P_msrA::lacZ chromosomal fusion reporter expressed in the indicated strains under stress conditions. (E) Quantitative RT-PCR analyses of msrA expression in the indicated strains under stress conditions. (F) EMSA was carried out to analyze interactions between His₆-SigH and the msrA promoter. The increasing amounts of His₆-SigH used were 0, 0.5, 1.0, 1.5, 2.5, and 3.5 μg. As negative controls, a 240-bp fragment from the msrA coding open reading frame instead of the msrA promoter (lane 8) and BSA instead of His₆-SigH (lane 7) were included in the binding assays. , free DNA; , major DNA-protein complex. Mean values with standard deviations (SD) (error bars) from at least three repeats are shown. *, P ≤ 0.05; **, P ≤ 0.01.