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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01rv042x00n
Title: Building a membrane: β-barrel outer membrane protein assembly in Escherichia coli
Authors: Hart, Elizabeth
Advisors: Silhavy, Thomas J
Contributors: Molecular Biology Department
Keywords: BAM complex
Escherichia coli
Outer membrane biogenesis
β-barrel outer membrane proteins
Subjects: Molecular biology
Microbiology
Genetics
Issue Date: 2020
Publisher: Princeton, NJ : Princeton University
Abstract: Gram-negative bacteria are a class of microbes with the distinguishing structure of an outer membrane (OM) in the cell envelope. The OM performs the essential function of selective permeability that allows vital nutrients to enter the cell but prevents toxic molecules and hydrophobic compounds from entering, many of which are detergents and antibiotics. Thus, the OM is a fundamental barrier to the clinical treatment of Gram-negative pathogens. The OM is a protein-rich bilayer that contains integral β-barrel outer membrane proteins (OMPs). OMPs are responsible for building and maintaining the integrity of the membrane as well as establishing the selective permeability of the OM. To reach the OM, OMPs must cross each layer of the cell envelope before being folded into the OM. Briefly, after translation in the cytoplasm, OMPs are translocated across the inner membrane (IM) by the Sec translocon. Chaperones bind the unfolded OMP at the periplasmic face of the IM and transport the substrate across the periplasm. The OMP substrate is delivered to the β-barrel assembly machine (BAM complex) for assembly and folding into the OM. As OMPs are critical for cell viability, the process of OMP assembly is closely monitored and tightly regulated by envelope stress responses. Here, we present a detailed investigation of OMP assembly by the BAM complex using suppressor analysis and well-characterized assembly-defective conditions. Our results demonstrate that envelope stress responses exquisitely monitor the OMP assembly process and that finely tuning activation of these pathways can have drastic implications on cell viability. Furthermore, this study has uncovered specific and non-redundant roles for the proteins of the BAM complex and has shown that these proteins largely work to regulate the activity of BamA, the essential BAM complex component responsible for the catalytic role of OMP assembly. In collaboration with Merck Pharmaceuticals, we have used this knowledge to identify and characterize a novel small molecule antibiotic that kills a broad spectrum of gram-negative pathogens through inhibition of OMP assembly by the BAM complex. This study sheds light on the mechanisms through which gram-negative bacteria regulate OMP assembly and establishes these processes as viable targets for antibiotic development.
URI: http://arks.princeton.edu/ark:/88435/dsp01rv042x00n
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Molecular Biology

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