Fig 4.

Altered responsiveness of A. baumannii PmrB motif sequence mutants to low pH and metal ions. (A) A schematic diagram of the amino acid sequence in the PmrB periplasmic region of A. baumannii is presented. Mutant strains with alanine substitutions for glutamate in the ExxE and ExxxE motifs were generated and transfected with a reporter plasmid. These were then incubated at pH 7.0 or 5.8 (B), and β-galactosidase activity was measured after incubation with 500 µM NaCl, KCl, MgCl₂, CaCl₂, FeSO₄, FeCl₃, ZnSO₄, AlCl₃, NiSO₄, Co(NO₃)₂, MnCl₂, or CuSO₄ for 60 min (C). The values are presented as relative values, with those at pH 7.0 or without metal ions set at 1.0. All experiments were performed in triplicate. Mean values and error bars represent standard deviations. Statistical significance is shown as a reference of the ATCC 19606 (WT) strain value using Dunnett’s test; ***P < 0.001. (D) The WT and PmrB (131AxxxA) strains in the logarithmic growth phase were exposed to 500 µM ZnSO₄ for 2 h. Subsequently, the bacterial suspension was adjusted to an OD₆₀₀ of 0.2. After incubating the bacterial suspension with 7 µg/mL polymyxin B or without antimicrobial agents for 2 h, 10 µL of the 10-fold serial dilution of the bacterial suspension was plated onto LB agar plates, which were then incubated at 37°C for 24 h. Experiments were conducted in triplicate, and images of representative agar plates are shown.