title: Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.
abstract: The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.
authors.affiliation: Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO 63110, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Saulino
authors.forename: D G
authors.initials: DG
authors.lastname: Thanassi
authors.forename: J S
authors.initials: JS
authors.lastname: Pinkner
authors.forename: S J
authors.initials: SJ
authors.lastname: Hultgren
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018830:Adhesins, Escherichia coli / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D033903:Periplasmic Proteins; D018272:Porins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D018830:Adhesins, Escherichia coli; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C084330:PapD protein, E coli; C092426:PapG protein, E coli; D033903:Periplasmic Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C111243:atpG protein, E coli; C489721:fimC protein, E coli; C489283:fimD protein, E coli; C063629:fimD protein, bacteria; C058769:fimH protein, E coli; C063055:fimbrillin; D036781:Fimbriae Proteins; D010450:Endopeptidases; C067127:prepilin peptidase protein, Bacteria; D014357:Trypsin