Sequential and spatially restricted interactions of assembly factors with an autotransporter β domain

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular (passenger) domain and a C-terminal beta barrel domain (beta 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 beta domain is integrated into the outer membrane and the relationship between beta domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the beta barrel assembly machinery (Bam) complex interact with the beta 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 beta domain, BamA, BamB, and BamD subsequently interacted with discrete beta domain regions. BamB and BamD remained bound to the beta domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of beta 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 beta barrel proteins.