Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.

Bacteria like Escherichia coli and Pseudomonas aeruginosa expel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a ␤-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous ␤-roll, ␤-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter-adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drugbinding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin-TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA 3-AcrB3-TolC3 model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.antibiotic resistance ͉ docked model ͉ membrane complex ͉ TolC exit duct

The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner-membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer-membrane (OM) porin-like TolC. These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross-linked in vivo complexes were isolated before and after substrate engagement. HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate. Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components. The IM-OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC. The bridge was transient, components reverting to IM and OM states after translocation. Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel. A similar picture was evident when the HlyD C-terminus was masked. Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate-induced, reversible bridging of the IM translocase to the OM export pore. Keywords: bacterial secretion mechanism/mitochondrial import/in vivo protein translocation/TolC membrane pore/traffic ATPase

Salmonella causes severe gastroenteritis in humans, entering non-phagocytic cells to initiate intracellular replication. Bacterial engulfment occurs by macropinocytosis, which is dependent upon nucleation of host cell actin polymerization and condensation ('bundling') of actin filaments into cables. This is stimulated by contact-induced delivery of an array of bacterial effector proteins, including the four Sips (Salmonella invasion proteins). Here we show in vitro that SipC bundles actin filaments independently of host cell components, a previously unknown pathogen activity. Bundling is directed by the SipC N-terminal domain, while additionally the C-terminal domain nucleates actin polymerization, an activity so far known only in eukaryotic proteins. The ability of SipC to cause actin condensation and cytoskeletal rearrangements was confirmed in vivo by microinjection into cultured cells, although as SipC associates with lipid bilayers it is possible that these activities are normally directed from the host cell membrane. The data suggest a novel mechanism by which a pathogen directly modulates the cytoskeletal architecture of mammalian target cells.

The bacterial TolC protein plays a common role in the expulsion of diverse molecules, which include protein toxins and antibacterial drugs, from the cell. TolC is a trimeric 12-stranded alpha/beta barrel, comprising an alpha-helical trans-periplasmic tunnel embedded in the outer membrane by a contiguous beta-barrel channel. This structure establishes a 140 A long single pore fundamentally different to other membrane proteins and presents an exit duct to substrates, large and small, engaged at specific inner membrane translocases. TolC is open to the outside medium but is closed at its periplasmic entrance. When TolC is recruited by a substrate-laden translocase, the entrance is opened to allow substrate passage through a contiguous machinery spanning the entire cell envelope, from the cytosol to the external environment. Transition to the transient open state is achieved by an iris-like mechanism in which entrance alpha-helices undergo an untwisting realignment, thought to be stabilized by interaction with periplasmic helices of the translocase. TolC family proteins are ubiquitous among gram-negative bacteria, and the conserved entrance aperture presents a possible cheomotherapeutic target in multidrug-resistant pathogens.