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STRUCTURAL STUDIES ON THE BIOGENESIS OF OMPS BY THE β-BARREL ASSEMBLY MACHINERY IN E. COLI

thesis
posted on 22.03.2022, 14:52 by Runrun WuRunrun Wu

The β-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a β-barrel fold which can vary from 8 to 36 β-strands, with each serving an important role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis has remained elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. Here we present our recent work on the structures of BAM in its near native environment, characterized by cryo-EM, and study its interaction with OMP substrates. Specifically, we focused on the role of BAM-mediated EspP biogenesis, and structurally characterized crosslinked intermediates to atomic resolution, allowing for a more complete understanding of BAM-mediated OMP biogenesis. We also characterized BAM-mediated OmpT and OmpA biogenesis, which further supports a BamA-budding model for OMP biogenesis. Given its essential role in Gram-negative bacteria, BAM is an attractive target for antibiotics, and we contributed to characterizing a novel antibiotic designed against BAM called darobactin, which binds to the lateral gate of BAM, thereby disrupting OMP biogenesis and leading to programmed bacterial lysis.

History

Degree Type

Doctor of Philosophy

Department

Biological Sciences

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Nicholas Noinaj

Additional Committee Member 2

Cynthia Stauffacher

Additional Committee Member 3

Leifu Chang

Additional Committee Member 4

Yun Zhou