Structural constraints of pyocin S2 import through the ferripyoverdine receptor FpvAI.

TonB-dependent transporters (TBDTs) mediate energized transport of essential nutrients into gram-negative bacteria. TBDTs are increasingly being exploited for the delivery of antibiotics to drug-resistant bacteria. While much is known about ground state complexes of TBDTs, few details have emerged about the transport process itself. In this study, we exploit bacteriocin parasitization of a TBDT to probe the mechanics of transport. Previous work has shown that the N-terminal domain of Pseudomonas aeruginosa-specific bacteriocin pyocin S2 (PyoS2NTD) is imported through the pyoverdine receptor FpvAI. PyoS2NTD transport follows the opening of a proton-motive force-dependent pore through FpvAI and the delivery of its own TonB box that engages TonB. We use molecular models and simulations to formulate a complete translocation pathway for PyoS2NTD that we validate using protein engineering and cytotoxicity measurements. We show that following partial removal of the FpvAI plug domain which occludes the channel, the pyocin's N-terminus enters the channel by electrostatic steering and ratchets to the periplasm. Application of force, mimicking that exerted by TonB, leads to unraveling of PyoS2NTD as it squeezes through the channel. Remarkably, while some parts of PyoS2NTD must unfold, complete unfolding is not required for transport, a result we confirmed by disulfide bond engineering. Moreover, the section of the FpvAI plug that remains embedded in the channel appears to serve as a buttress against which PyoS2NTD is pushed to destabilize the domain. Our study reveals the limits of structural deformation that accompanies import through a TBDT and the role the TBDT itself plays in accommodating transport.

Lipids mediate supramolecular outer membrane protein assembly in bacteria.

ß Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric ß barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.