The S-layer is an exoskeleton-like structure that imparts mechanical and osmotic stabilization to the cell wall.

Surface layers (S-layers) are 2D paracrystalline protein monolayers covering the cell envelope of many prokaryotes and archaea. Proposed functions include a role in cell support, as scaffolding structure, as molecular sieve, or as virulence factor. holds two S-layers, composed of Sap or EA1, which interchange in early and late exponential growth phase. We previously found that acute disruption of Sap S-layer integrity, by means of nanobodies, results in severe morphological cell surface defects and cell collapse. Remarkably, this loss of function is due to the destruction of the Sap lattice structure rather than detachment of monomers from the cell surface. Here, we combine force nanoscopy and light microscopy observations to probe the contribution of the S-layer to the mechanical, structural, and functional properties of the cell envelope, which have been so far elusive. Our experiments reveal that cells with a compromised S-layer lattice show a decreased compressive stiffness and elastic modulus. Furthermore, we find that S-layer integrity is required to resist cell turgor under hypotonic conditions. These results present compelling experimental evidence indicating that the S-layers can serve as prokaryotic exoskeletons that support the cell wall in conferring rigidity and mechanical stability to bacterial cells.