Probing the dynamics, orientation and conformation of lipopolysaccharide in bacterial membranes through bioorthogonal chemistry and single-molecule bi

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The outer membrane (OM) of Gram-negative bacteria forms a significant barrier that helps to protect bacteria from their environment and increases resistance to several antibiotics. The OM is an asymmetric bilayer composed of an inner leaflet of phospholipid and an outer leaflet of lipopolysaccharide (LPS) with OM proteins (OMPs) spanning the bilayer. Recently, we demonstrated that OMPs are turned over via a method termed binary partitioning. New OMPs were shown to be inserted preferentially at the mid-cell with old OMPs pushed to the poles during cell elongation, facilitating fast adaptation to a new environmental niche at the population-level.The spatio-temporal dynamics of LPS in the OM have remained a controversial topic since the introduction of the fluid mosaic model in the 1970s. We propose to finally unequivocally establish the lateral diffusion properties of LPS, explore its potential co-localisation with OMPs, and determine the conformation and orientation of its polysaccharide head group relative to the extracellular surface. This project is wholly reliant upon an interdisciplinary collaboration across the fields of glycoscience, bioorthogonal chemistry and single-molecule microbial biophysics.Using an interdisciplinary approach combining bioorthogonal click-labelling of glycans and single-molecule fluorescence microscopy, we aim to (1) explore the underpinning mechanism governing the spatio-temporal dynamics of LPS through chemical bioconjugation of the LPS insertion machinery in the OM, and simultaneous click-labelling of Kdo sugars in LPS, for fluorescence co-localisation microscopy; and (2) probe the conformation of LPS in the membrane through the dual metabolic labelling of both glycans and acetate groups, and single-molecule FRET experiments.