Investigating Cell Wall Permeability in Bacillus subtilis

The Gram-positive cell wall functions as a major load-bearing structure, and here we aim to characterize whether it also functions as a major permeability barrier. The outermost layer of the Gram-positive cell envelope, a thick peptidoglycan cell wall, may function as a permeability barrier analogous to the outer membrane of Gram-negative bacteria. However, its effectiveness as such a barrier remains to be fully characterized. By using flow cytometry and single-cell microfluidics assays to measure the diffusion of novel genetically encoded fluorescent probes through the Bacillus subtilis cell wall, we found that it is essentially impermeable to proteins 15 kDa or larger. Inducing autolysis (programmed digestion of peptidoglycan) permeabilized the cell wall, whereas de-energizing the cell slowed diffusion through the cell wall. These data demonstrate that proteins traverse the cell wall through transient openings during peptidoglycan remodeling, not passive diffusion through fixed pores.