Probing the spatio-temporal dynamics of lipopolysaccharide in the Gram-negative bacterial outer membrane

The Gram-negative bacterial outer membrane (OM) is an asymmetric bilayer with an inner leaflet composed of phospholipid and an outer leaflet of lipopolysaccharide (LPS). The OM forms an impermeable barrier to environmental challenges including many commonly used antibiotics. It is known that OM proteins (OMPs) do not diffuse freely within the OM. Newly inserted OM proteins are inserted at mid-cell, pushing old OM proteins to the poles with cell growth. By contrast little is known about the insertion pattern of LPS. Previous studies of LPS lateral diffusion are contradictory with both mobile and immobile behaviour observed. We have investigated the spatio-temporal dynamics of LPS using fluorescence microscopy techniques. LPS is fluorescently labeled using a metabolic labeling technique to incorporate a sugar analogue into LPS, which can be fluorescently labeled with a specific bio-orthogonal click-it reaction. Using both fluorescence recovery after photobleaching and single-molecule tracking experiments we show that LPS is unable to diffuse laterally in the OM. The factors on which this immobility depends are explored and it is shown that the immobility is independent of LPS polysaccharide structural complexity and only partially dependent on metal ion mediated LPS-LPS interactions. Using pulse-labeling methods it is shown that there is a preference for insertion of new LPS at mid-cell, as was observed for OMPs. Midcell incorporation of new material combined with the lack of diffusion in the OM leads to a mechanism by which OM material may be rapidly turned over in response to environmental changes. To further explore the parameters which control the insertion of new material into the OM a model for OM growth is developed. Preliminary results suggest that targeting of new protein and LPS insertion complexes to mid-cell is sufficient to reproduce the experimentally observed insertion of new material at mid-cell