Supra-molecular organization of the pyoverdine bio-synthetic pathway in Pseudomonas aeruginosa

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.