Fig. 5: FprB enhances the survival of P. aeruginosa by inhibiting Ga(NO₃)₃-induced intracellular iron and ROS (reactive oxygen species) accumulation.

a Intracellular total iron and Fe²⁺ accumulation. Bacteria were cultured for 6 h in LB broth with or without Ga(NO₃)₃, starting at an OD600 of 0.05. Total iron or Fe²⁺ were measured as described in Methods, and normalized to OD600. b, c ROS and H₂O₂ accumulation in bacteria grown for 4 h in the absence or presence of Ga(NO₃)₃, detected by flow cytometry using carboxy-H2DCFDA and Peroxy Orange-1, respectively. d, e Plate spot assays for sensitivity of PAO1 wt, ∆fprB and ∆fprB-Com strains to H₂O₂ and Plumbagin. All strains were initially adjusted to an OD600 of 0.003 and then serially diluted tenfold before spotting onto LB agar plates (10⁻² to 10⁻⁶). f Volcano plot of differential expression between ∆fprB and wt transcriptomes. Statistical analysis was performed using a two-sided Wald test implemented in DESeq2, with Benjamini-Hochberg correction for multiple comparisons. Significant difference was defined as (|log₂FC|> 2 with Padj < 0.05). Red (upregulated); cyan (downregulated), grey (no difference). g The role of downregulated NO reductase (NorBC), N₂O reductases (NosZ) and NosR, and upregulated PA2691 and CyoABCDE in bacterial aerobic respiration. h NO levels in the supernatant of bacterial cultures. i Proposed mechanism of gallium-induced cell death in wt and ∆fprB mutant. Statistical analysis for (a, c, and h) was performed using Two-way ANOVA and Tukey’s multiple-comparison test. All data are presented as mean ± SD from three biological replicates. Source data are provided as a Source Data file.