Pseudomonas aeruginosa TfpW is a multifunctional D-Araf glycosyltransferase and oligosaccharyltransferase

ABSTRACT TfpW is an oligosaccharyltransferase that modifies the subunits of type IV pili from group IV strains of Pseudomonas aeruginosa with oligomers of α-1,5-linked-D-arabinofuranose (D-Ara f ). Besides its oligosaccharyltransferase activity, TfpW may be responsible for periplasmic translocation and polymerization of D-Ara f . Here we investigated these potential roles of TfpW in Pa5196 pilin glycosylation. Topology studies confirmed the periplasmic location of loop 1 and the large C-terminus domain, however the central portion of TfpW had an indeterminate configuration. Reconstitution of the Pa5196 pilin glycosylation system by providing pilA , tfpW +/- tfpX and the D-Ara f biosynthesis genes PsPA7_6246-6249 showed that TfpW is sufficient for glycan polymerization and transfer to pilins in P. aeruginosa PAO1, while TfpX is also necessary in Escherichia coli . In addition to PsPA7 _ 6246, DprE1 (PsPA7 _ 6248) and DprE2 (PsPA7 _ 6249), the GtrA-like component PsPA7 _ 6247 was required for pilin glycosylation in E. coli versus PAO1. In a PAO1 Δ arnE/F mutant, loss of PsPA7_6247 expression decreased the level of pilin glycosylation, suggesting that arnE/F may play a role in pilin glycosylation when PsPA7_6247 is absent. Bacterial two-hybrid studies showed interactions of TfpW with itself, TfpX, PsPA7 _ 6247 and DprE2, suggesting the formation of a complex that enables efficient pilin glycosylation. Fluorescence microscopy of E. coli and Pa5196 ΔdprE1 expressing a DprE1-sGFP fusion showed that the protein is expressed in the cytoplasm, supporting our model that includes cytoplasmic biosynthesis of the lipid carrier-linked D-Ara f precursor prior to its periplasmic translocation. Together these data suggest that TfpW may be the first example of a trifunctional flippase, glycosyltransferase, and oligosaccharyltransferase.