title: Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion.
abstract: BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Browning
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Vassiliy N
authors.initials: VN
authors.lastname: Bavro
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Mason
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Yanina R
authors.initials: YR
authors.lastname: Sevastsyanovich
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Rossiter
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Jeeves
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Wells
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Knowles
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Adam F
authors.initials: AF
authors.lastname: Cunningham
authors.affiliation: College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
authors.forename: James W
authors.initials: JW
authors.lastname: Donald
authors.affiliation: College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Overduin
authors.affiliation: Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Henderson
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002678:Chimera / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins