The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane
Sachith D. Gunasinghe,
Takuya Shiota,
Christopher J. Stubenrauch,
Keith E. Schulze,
Chaille T. Webb,
Alex J. Fulcher,
Rhys A. Dunstan,
Iain D. Hay,
Thomas Naderer,
Donna R. Whelan,
Toby D.M. Bell,
Kirstin D. Elgass,
Richard A. Strugnell,
Trevor Lithgow
Affiliations
Sachith D. Gunasinghe
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Takuya Shiota
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Christopher J. Stubenrauch
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Keith E. Schulze
Monash Micro Imaging, Monash University, Clayton, VIC 3800, Australia
Chaille T. Webb
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Alex J. Fulcher
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Rhys A. Dunstan
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Iain D. Hay
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
Thomas Naderer
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800, Australia
Donna R. Whelan
School of Chemistry, Monash University, Clayton, VIC 3800, Australia
Toby D.M. Bell
School of Chemistry, Monash University, Clayton, VIC 3800, Australia
Kirstin D. Elgass
Monash Micro Imaging, Monash University, Clayton, VIC 3800, Australia
Richard A. Strugnell
Department of Microbiology & Immunology, University of Melbourne, Parkville, VIC 3052, Australia
Trevor Lithgow
Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.
