title: Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.
abstract: 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.
authors.affiliation: Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Ieva
authors.forename: Pu
authors.initials: P
authors.lastname: Tian
authors.forename: Janine H
authors.initials: JH
authors.lastname: Peterson
authors.forename: Harris D
authors.initials: HD
authors.lastname: Bernstein
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D008074:Lipoproteins / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; C557407:BamB protein, E coli; C513739:BamD protein, E coli; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; C072721:Skp protein, E coli; C107189:EspP protein, E coli; D012697:Serine Endopeptidases; C490900:SurA protein, E coli; D019696:Peptidylprolyl Isomerase