The assembly of the autotransporter family of bacterial outer membrane proteins

The Bernstein lab is investigating the assembly of proteins that reside in the outer membrane of Gram-negative bacteria. While most integral membrane proteins contain one or more α-helical membrane spanning segments, bacterial outer membrane proteins are anchored to the membrane by a single amphipathic β sheet that folds into a distinctive closed cylindrical structure called a “β barrel”. Previous studies have shown that a conserved heteroligomer (the barrel assembly machinery or “Bam” complex) catalyzes the integration of β barrels into the outer membrane, but the mechanism by which it promotes insertion is unknown. In many of our experiments we have focused on the assembly of a specific class of outer membrane proteins produced by pathogenic bacteria called “autotransporters”. These proteins contain a large N-terminal extracellular domain ("passenger domain”) in addition to a C-terminal β barrel. The passenger domains of individual members of the autotransporter superfamily play a variety of roles in pathogenesis; in some cases they function as adhesins, but in other cases they are cleaved from the cell surface and function as soluble virulence factors. We have obtained evidence that the β barrel of a model autotransporter produced by E. coli O157:H7 (EspP) begins to fold in the periplasm (the space between the two cell membranes) and then binds to several subunits of the Bam complex in a stereospecific fashion. We have also found that the Bam complex catalyzes the assembly of the EspP β barrel in two separable binding and integration steps. Finally, although it was proposed many years ago that the passenger domain is secreted through a channel formed by the covalently linked β barrel (whence the name “autotransporter”), our work has strongly challenged this hypothesis by indicating that the Bam complex plays a key role in the secretion reaction.