Analysis of Surface Protein Expression Reveals the Growth Pattern of the Gram-Negative Outer Membrane

The outer membrane (OM) of Gram-negative bacteria is a complex bilayer composed of proteins, phospholipids, lipoproteins, and lipopolysaccharides. Despite recent advances revealing the molecular pathways underlying protein and lipopolysaccharide incorporation into the OM, the spatial distribution and dynamic regulation of these processes remain poorly understood. Here, we used sequence-specific fluorescent labeling to map the incorporation patterns of an OM-porin protein, LamB, by labeling proteins only after epitope exposure on the cell surface. Newly synthesized LamB appeared in discrete puncta, rather than evenly distributed over the cell surface. Further growth of bacteria after labeling resulted in divergence of labeled LamB puncta, consistent with a spatial pattern of OM growth in which new, unlabeled material was also inserted in patches. At the poles, puncta remained relatively stationary through several rounds of division, a salient characteristic of the OM protein population as a whole. We propose a biophysical model of growth in which patches of new OM material are added in discrete bursts that evolve in time according to Stokes flow and are randomly distributed over the cell surface. Simulations based on this model demonstrate that our experimental observations are consistent with a bursty insertion pattern without spatial bias across the cylindrical cell surface, with approximately one burst of ∼10−2 µm2 of OM material per two minutes per µm2. Growth by insertion of discrete patches suggests that stochasticity plays a major role in patterning and material organization in the OM. Author Summary: All Gram-negative bacteria share common structural features, including an inner membrane, a stiff cell wall, and an outer membrane. Balancing growth in all three of these layers is critical for bacterial proliferation and survival, and malfunctions in growth often lead to cellular deformations and/or cell death. However, relatively little is known about how the incorporation of new material into the outer membrane is regulated in space and time. This work combines time-lapse microscopy with biophysical modeling and simulations to examine potential mechanisms by which new material is added to the outer membrane of the rod-shaped Gram-negative bacterium Escherichia coli. Our results indicate that the outer membrane grows in discrete bursts randomly distributed over the cylindrical cell surface. Each insertion event adds a random amount of new material, pushing old material into new locations and thus expanding the cell membrane. Using our biophysical model, we generated simulated fluorescence images and directly compared analyses of our experimental and computational results to constrain the rate and size of bursts of growth. Together, this indicates that growth of the outer membrane does not require spatial regulation, and the stochastic nature of insertion may contribute to the establishment of cellular patterning and asymmetry.