title: Mutational and topological analysis of the Escherichia coli BamA protein.
abstract: The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Browning
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Sophie A
authors.initials: SA
authors.lastname: Matthews
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Rossiter
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Yanina R
authors.initials: YR
authors.lastname: Sevastsyanovich
authors.affiliation: School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Jeeves
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Mason
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Wells
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Wardius
authors.affiliation: School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Knowles
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Adam F
authors.initials: AF
authors.lastname: Cunningham
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Vassiliy N
authors.initials: VN
authors.lastname: Bavro
authors.affiliation: School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Overduin
authors.affiliation: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Henderson
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins