P62: Role of beta barrel assembly in virulence factor secretion

Autotransporters are a family of virulence factors widely distributed among Gram-negative bacteria. They consist of an N-terminal extracellular domain (“passenger domain”) and a C-terminal beta barrel domain (“beta domain”) that anchors the protein in the outer membrane (OM). Despite significant effort, the mechanism of passenger domain secretion has remained elusive. It has been known for many years that the beta domain is required for secretion, but its exact role is unclear. To gain insight into the function of the beta domain we studied two mutants of EspP, a model autotransporter produced by E. coli O157:H7. Experiments that assessed the sensitivity of the beta domain to SDS denaturation and monitored the time course of passenger domain secretion showed that the mutations reduce beta barrel stability and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the G1066A and G1081D mutations do not affect the initial binding of the beta domain to the factor that catalyzes the insertion of beta barrel proteins into the OM (the so-called “Bam complex”), but rather slow down the subsequent membrane insertion reaction itself. These results suggest that the beta domain plays a more direct role in the translocation reaction than targeting of the passenger domain to the OM. Our data also provides significant insight into the function of the Bam complex which has implications for understanding OM protein assembly in mitochondria and chloroplasts.