title: Reconstitution of bacterial autotransporter assembly using purified components.
abstract: Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular ('passenger') domain and a C-terminal β barrel ('β') domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro. Our results indicate that the membrane integration of the β domain is the rate-limiting step in autotransporter assembly and that passenger domain translocation does not require the input of external energy. Furthermore, experiments using nanodiscs strongly suggest that autotransporter assembly is catalyzed by a single copy of the Bam complex. Finally, we describe a method to purify a highly active form of the Bam complex that should facilitate the elucidation of its function.
authors.affiliation: Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States.
authors.forename: Giselle
authors.initials: G
authors.lastname: Roman-Hernandez
authors.affiliation: Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States.
authors.forename: Janine H
authors.initials: JH
authors.lastname: Peterson
authors.affiliation: Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States.
authors.forename: Harris D
authors.initials: HD
authors.lastname: Bernstein
mesh_terms: D055162:Biocatalysis; D019453:Escherichia coli O157 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D011510:Proteolipids; D037521:Virulence Factors; C020485:proteoliposomes
keywords: E. coli; biochemistry; infectious disease; membrane proteins; microbiology; protein folding; protein translocation; virulence factors