Figure 5.

The sensitivity of P. aeruginosa to GaPPIX depends on the expression of heme-uptake systems. (A) Approximately 5 × 10⁶ bacterial cells were seeded on DCAA agar plates containing 200 μg/ml Cb, then disks soaked with 10 μl of a 15 mM solution of GaPPIX were deposited on the agar surface. Plates were incubated for 16 h at 37°C. (B) Growth of the P. aeruginosa ΔhasRΔphuR mutant strain carrying the empty vector pUCP18 (white bars), or overexpressing hasR from plasmid pUCPhasR (light gray bars) or phuR from plasmid pUCPphuR (dark gray bars), or both hasR and phuR from plasmid pUCPhasRphuR (black bars), in DCAA supplemented with increasing concentrations of GaPPIX for 24 h at 37°C. (C) SDS-PAGE analysis of the outer membrane proteins of different P. aeruginosa PAO1 strains. Strains and plasmids are indicated on the top of each lane. M is the molecular mass marker (kDa), with band sizes on the left. The position of the putative 82 kDa PhuR and 94 kDa HasR outer membrane receptors is indicated by arrows on the right. (D) Rescue effect of hemin (Hm) or hemoglobin (Hb) from GaPPIX growth inhibition in the ΔhasRΔphuR mutant strain overexpressing either phu from plasmid pUCPphuR or hasR from plasmid pUCPhasR. Approximately 5 × 10⁶ bacterial cells were seeded on DCAA agar plates containing 200 μM Cb. Blank discs were soaked with 10 μl of a 15 mM solution of GaPPIX or with 75 μg of either Hm or Hb. Plates were incubated at 37°C for 16 h. (E) Growth of the P. aeruginosa ΔpvdAΔpchD mutant strain carrying the empty vector pUCP18 (black bars), or overexpressing both hasR and phuR from plasmid pUCPhasRphuR (white bars), in DCAA supplemented with increasing concentrations of GaPPIX for 24 h at 37°C. Values are the mean of two independent experiments, each one performed in triplicate ± the standard deviation. Images in B and D are representative of two independent experiments yielding similar results.