Fig 3.

Identification of PmrAB response factors in A. baumannii. (A) A schematic diagram of the PmrAB reporter assay in A. baumannii. Upon activation of PmrB, PmrA is phosphorylated and binds to the upstream region of the KAMO5_07940 gene, which induces lacZ expression and produces β-galactosidase. The β-galactosidase activity was evaluated after incubation at pH 7.0 or 5.8 (B) or with 500 µM NaCl, KCl, MgCl₂, CaCl₂, FeSO₄, FeCl₃, ZnSO₄, AlCl₃, NiSO₄, Co(NO₃)₂, MnCl₂, or CuSO₄ (C) for 60 min. The values are expressed as relative values, with those at pH 7.0 or without metal ions set at 1.0. (D) Relative β-galactosidase activity is shown after 60 min of incubation with Fe²⁺ (FeSO₄) or Fe³⁺ (FeCl₃) at concentrations of 0, 100, 500, or 1,000 µM for the ATCC 19606 strain harboring the reporter plasmid. (E) β-galactosidase activity was evaluated after 60 min of incubation of the ATCC 19606 strain transfected with the reporter plasmid with or without 1,000 µM of FeCl₃, and with 0, 0.5, 1, or 2 mM of ascorbic acid used as a reducing agent. The values are shown as relative values, with the value without FeCl₃ addition set at 1.0. All experiments were performed in triplicate. Mean values and error bars represent standard deviations. Statistical significance is shown using Tukey’s test as a reference of pH 7.0 or untreated values (B, C), unpaired t-test compares Fe²⁺ and Fe³⁺ (D), and Dunnett’s test as a reference of the untreated value (E); ***P < 0.001, **P < 0.01, *P < 0.05.