The outer membrane (OM) is a formidable barrier that has made antibiotic drug discovery in Gram-negatives exceedingly difficult. Many antibiotics which are effective against Gram-positive bacteria cannot permeate the Gram-negative OM to reach their intracellular targets. Thus, it is important to explore unconventional approaches to overcome the intrinsic resistance conferred by the OM. Herein, we used both genetic and chemical means to compromise OM integrity to potentiate the activity of large-scaffold antibiotics against Escherichia coli. First, we mapped the genetic interaction network of OM biosynthetic genes using synthetic genetic arrays (SGAs) to reveal permeability determinants of the E. coli OM. This led to the creation of a publicly accessible dataset of ~155,400 double deletion strains with growth data in the presence of the large-scaffold antibiotics rifampicin and vancomycin. Investigations of a subset of synthetic sick interactions revealed connectivity in the context of permeability between lipopolysaccharide (LPS) inner core biosynthetic genes and an enigmatic gene involved in enterobacterial common antigen (ECA) regulation. Second, we leveraged a chemical screening platform based on the observation that disruption of the E. coli OM leads to antagonism of vancomycin activity at cold temperatures to uncover molecules that potentiate Gram-positive-targeting antibiotics at 37 ºC. Two of these compounds, liproxstatin-1 and MAC-0568743, were characterized to bind to LPS and disrupt OM integrity specifically without impacting the inner membrane (IM). Third, we performed genetic and chemical screening to unearth targets capable of potentiating the activity of Gram-positive-targeting antibiotics against E. coli. This validated the OM as a valuable target for antibiotic adjuvants and led to the discovery of two membrane active compounds and an inhibitor of lipid A biosynthesis. Overall, this thesis emphasizes the importance of elucidating biological factors contributing to OM permeability and the attractiveness of the OM as a target for antibiotic potentiators. / Thesis / Doctor of Philosophy (PhD)

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