Fig. 5.

Analysis of the changes in the expression (panel A) and the transcription (panels B, C, and D) of genes involved in iron-uptake pathways in P. aeruginosa cells grown under iron-limited conditions (CAA medium) in the absence or presence of exosiderophores. A, Proteomic analyses were performed on P. aeruginosa PAO1 cells grown over night in CAA supplemented, or not, with 10 μm FERRI, VIB, or YER. Average values measured in CAA in the absence of any supplementation with siderophores were plotted against average values measured in CAA supplemented with either 10 μm FERRI, VIB, or YER. Median values represent the median of the relative intensity of each protein, normalized against all proteins detected by shotgun analysis (n = 3). Supplemental Data S2 shows the detailed results of protein identification and quantitation. B, fptA and fpvA encode the TBDTs of PCH and PVD, respectively, pfeA of ENT, fvbA of VIB, femA of mycobactin and carboxymycobatin, fiuA of FERRI, foxA of ferrioxamine B and PA0434 of a hypothetical TBDT. RT-qPCR was performed on P. aeruginosa PAO1 cells grown in CAA medium during 8 h as described in Materials and Methods, with or without 10 μm ENT, FERRI, VIB, or YER. The data were normalized relative to the reference gene uvrD and are representative of three independent experiments performed in triplicate (n = 3). Results are given as the ratio between the values obtained in the presence of siderophores over those obtained in their absence. C, RT-qPCR analyses were performed on P. aeruginosa PAO1 cells grown in CAA during 8 h as described in Materials and Methods and supplemented, or not, with a mixture of 2 μm ENT, 2 μm FERRI, 2 μm VIB, and 2 μm YER (total of 8 μm siderophores). The experiment was repeated with a mixture of 10 μm of each siderophore (total of 40 μm siderophores). As for panel B, the data were normalized relative to the reference gene uvrD and are representative of three independent experiments performed in triplicate (n = 3). Results are given as the ratio between the values obtained in the presence of the siderophores over those obtained in their absence. The data represent 3 independent experiments with 3 technical replicates. D, RT-qPCR was performed on P. aeruginosa PAO1 grown in CAA medium, with or without 2 μm ENT, 2 μm FERRI, 2 μm VIB, and 2 μm YER (total concentration of siderophores 8 μm) for 3 and 8 h. As for panel B, the data were normalized relative to the reference gene uvrD and are representative of three independent experiments performed in triplicate (n = 3). Results are given as the ratio between the values obtained in the presence of the siderophores over those obtained in their absence. The data represent 3 independent experiments with 3 technical replicates.