Cell wall damage increases macromolecular crowding effects in the cytoplasm.

The intracellular milieu is crowded with biomacromolecules. Macromolecular crowding changes the interactions, diffusion, and conformations of biomacromolecules. Changes in intracellular crowding have been mostly ascribed to differences in biomacromolecule concentration. However, spatial organization of these molecules should play a significant role in crowding effects. Here, we find that cell wall damage causes increased crowding effects in the cytoplasm. Using a genetically encoded macromolecular crowding sensor, we see that crowding effects in spheroplasts and penicillin-treated cells well surpass crowding effects obtained using hyperosmotic stress. The crowding increase is not because of osmotic pressure, cell shape, or volume changes and therefore not crowder concentration. Instead, a genetically encoded nucleic acid stain and a DNA stain show cytoplasmic mixing and nucleoid expansion, which could cause these increased crowding effects. Our data demonstrate that cell wall damage alters the biochemical organization in the cytoplasm and induces significant conformational changes in a probe protein.