The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.

The bio-synthesis of the pyoverdine siderophore (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of Non-Ribosomal Peptide Synthetases (NRPS). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesised by co-expressed accessory enzymes. It is generally accepted that different enzymes of a secondary metabolic pathway must organise into macro-molecular complexes. However, evidence for complexes like siderosomes in the cellular context are missing.Here, we used in vitro single-molecule tracking and FRET–FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed PvdA is physically interacting with PvdJ, PvdI, PvdL and PvdD, the four NRPS of the pyoverdine pathway. The binding modes of PvdA are strikingly different according to the NRPS it is interacting with suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS.Our data provide evidence for strongly organised multi-enzymatic complexes responsible for the bio-synthesis of PVD and suggest that finely controlled co-localisation of sequential enzymes seems to be required to promote metabolic efficiency.