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

Abstract

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.

Exploring protein–protein interactions with large differences in protein expression levels using FLIM-FRET

“…Bacterial strains, plasmids and growth conditions P. aeruginosa strains used in this study are described in (25). Bacteria were grown in 5 mL lysogeny broth (LB) (Difco) at 30 • C under 220 rpm orbital shaking.…”

“…Large differences in the protein expression levels of donors and acceptors are frequently observed in cells but are poorly reported as such in FLIM experiments. In a recent work, Gasser et al [25] characterized the interaction of PvdA with PvdJ in live Pseudomonas aeruginosa. PvdA is an ornithine hydroxylase [26] working in tandem with the hydroxy-ornithine transformylase PvdF [27] to generate fOH-Ornithine.…”

“…PvdI is a NRPS involved in the bio-synthesis of pyoverdine in PAO1 together with PvdJ, PvdL and PvdD [31,32]. As PvdJ, the expression level of PvdI is very low as compared to that of PvdA [25]. Both PvdI and PvdJ are responsible for the addition of a hydroxy-ornithine in the pyoverdine sequence.…”

Novel Insights on Pyoverdine: From Biosynthesis to Biotechnological Application

“…The fatty acid unit is then transferred to the second module of PvdL, catalysing the acylation of an L-glutamate residue. The next two PvdL modules promote the sequential incorporation of D-Tyrosine (D-Tyr) and L-2,4 Diaminobutyrate (L-Dab), thus forming a tetrahydropyrimidine ring, i.e., the precursor of the chromophore [ 43 , 59 ].…”