Bacterial protein import mediated by an iron transporter

Multidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand β-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2NTD) and FpvAI is presented, which shows pyoS2NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2NTD translocates through the FpvAI β-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.