Macromolecular organization of the pyoverdine biosynthetic pathway in Pseudomonas aeruginosa.

Abstract

The biosynthesis of the pyoverdine siderophore in Pseudomonas aeruginosa inlvolves multiple enzymes including Non-Ribosomal Peptide Synthetases (NRPS). We used in vitro single-molecule tracking and FRET-FLIM (Forster resonance energy transfer measured by fluorescence lifetime microscopy) to explore the spatial partitioning of the ornithine hydroxylase PvdA and to characterize its interactions with NRPS. The statistical description of thousands of single PvdA traces in cells using jump distance distribution analysis showed PvdA was mostly diffusing bound to large complex in the cytoplasm with a small exchangeable trapped fraction diffusing slower. FRET-FLIM clearly showed PvdA are physically interacting with the four NRPS PvdJ, PvdI, PvdL and PvdD of the pyoverdine pathway in the cellular context. Our data provide evidence for strongly organized multi-enzymatic biosynthetic complexes named siderosomes responsible for the siderophore biosynthesis. PvdA binding mode is strikingly different according to the NRPS it is interacting with suggesting PvdA binding site have co-evolved with the enzymatic active sites of NRPS.