Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal β barrel domain ("β domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the β domain is integrated into the outer membrane and the relationship between β domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the β barrel assembly machinery (Bam) complex interact with the β domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion. Although the chaperone Skp initially interacted with the entire β domain, BamA, BamB, and BamD subsequently interacted with discrete β domain regions. BamB and BamD remained bound to the β domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of β domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In addition to leading to a detailed model of autotransporter biogenesis, our results suggest that the lipoprotein components of the Bam complex play a direct role in the membrane integration of β barrel proteins.membrane protein assembly | molecular chaperones | protein translocation

Escherichia coli ͉ microbial pathogenesis ͉ protein folding ͉ protein translocation

SummaryBacterial autotransporters are proteins that contain a small C-terminal ' β β β β domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspP ∆ ∆ ∆ ∆ 1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspP ∆ ∆ ∆ ∆ 1-851 behaves as a compact monomer and strongly suggest that the channel formed by the β β β β domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspP ∆ ∆ ∆ ∆ 1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP β β β β domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.