(re)Defining the nature of the Gram-negative outer membrane

The impermeability of the outer membrane (OM) of Gram-negative bacteria contributes to organisms such as Escherichia coli and Pseudomonas aeruginosa dominating the WHO’s antibiotics resistance list. We discovered recently that contrary to decades-old assumptions, the OM is a spatiotemporally organised superstructure where outer membrane proteins and lipopolysaccharides come together to form integrated networks that span the bacterial surface. Not only do these networks create a robust, immobile, protective shield they also endow the membrane with specific biology, such as coordinating growth of the OM and cell wall. This proposal will uncover the structural principles underpinning these networks in non-pathogenic and pathogenic bacteria and the impact these networks have on antibiotic susceptibility. Our interdisciplinary team, which encompasses OM biophysics and imaging, proteomics, atomic force microscopy, chemical biology, cryo-electron microscopy/cryo-electron tomography and molecular dynamics simulations, aims to answer three questions: 1. What are the structural principles governing OM assembly? 2. How universal are principles of OM assembly? 3. How do polymyxin antibiotics exploit OM structure? Defining the molecular architecture of the OM will have far- reaching implications for understanding its unique biology and the interplay with the other layers of the cell envelope as well as accelerating rational design of membrane disrupting antibiotics.