Outer membrane crosslink density facilitates extracellular vesicle formation in E. coli*

Bacteria produce small spheres of membrane known as membrane vesicles. Vesicles are likely produced through several different mechanisms, including the outward blebbing of the membrane. Blebbing of the bacterial membrane involves both structural changes to the membrane and separation of the membrane from the cell wall. Gram-negative bacteria, such as E. coli, contain both inner and outer membranes attached to an internal cell wall via protein crosslinkers. Past experiments have shown removal of the most prevalent crosslinker protein leads to an increase in vesicle production. Here we examine how the relative production of vesicles scales with the number of crosslinks between the outer membrane and the cell wall. Building off of prior theoretical work concerning the ability of a membrane to squeeze through holes in a cell wall, we model how the size of membrane regions detached from the cell wall influence vesicle production. Predictions from the model were tested via experimental measurements of vesicle production in bacteria with variable expression of crosslinker proteins. These results explain how proteins that attach the outer membrane to the cell wall regulate vesicle production.