title: An In Vitro Assay for Substrate Translocation by FhaC in Liposomes.
abstract: The two-partner secretion (TPS) pathway is used by gram-negative bacteria to secrete a large family of virulence exoproteins. Its name is derived from the fact that it involves two proteins, a secreted TpsA protein and a cognate TpsB transporter in the outer membrane. A typical TPS system is represented by the filamentous hemagglutinin FhaB (TpsA protein) and its transporter FhaC (TpsB protein) of Bordetella pertussis. Results from mutational analysis and heterologous expression experiments suggested that FhaC is essential for FhaB translocation across the outer membrane of bacteria. We have devised a cell-free biochemical assay to reconstitute in vitro the translocation of FhaB into reconstituted membrane vesicles. Thereby the clearest evidence has been provided that the single β-barrel FhaC protein serves as the sole translocator to transport FhaB across the outer membrane. This is the first in vitro assay for protein secretion across the Escherichia coli outer membrane and the detailed protocol described here should be amenable to modifications and application to the analysis of related protein transport events occurring at the outer membranes of gram-negative bacteria.
authors.affiliation: Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, Stefan-Meier-Straße 17, Freiburg, 79104, Germany.
authors.forename: Enguo
authors.initials: E
authors.lastname: Fan
authors.affiliation: Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, Stefan-Meier-Straße 17, Freiburg, 79104, Germany.|Faculty of Biology, University of Freiburg, Freiburg, 79104, Germany.
authors.forename: Derrick
authors.initials: D
authors.lastname: Norell
authors.affiliation: Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, Stefan-Meier-Straße 17, Freiburg, 79104, Germany. matthias.mueller@biochemie.uni-freiburg.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Müller
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001886:Bordetella pertussis / Q000166:cytology / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D021381:Protein Transport; D011510:Proteolipids / Q000378:metabolism*; D012995:Solubility; D013104:Spheroplasts / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D011510:Proteolipids; D010566:Virulence Factors, Bordetella; C098052:fhaC protein, Bordetella pertussis; C065273:filamentous hemagglutinin adhesin, Bordetella pertussis; C020485:proteoliposomes
keywords: Bacterial outer membrane; Escherichia coli; Protein transport; Proteoliposome; Reconstitution; Spheroplasts; Two-partner secretion; β-barrel membrane proteins