We used a novel atomic force microscopy (AFM)-based technique to compare the local viscoelastic properties of individual gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacterial cells. We found that the viscoelastic properties of the bacterial cells are well described by a three-component mechanical model that combines an instantaneous elastic response and a delayed elastic response. These experiments have allowed us to investigate the relationship between the viscoelastic properties and the structure and composition of the cell envelope. In addition, this is the first report in which the mechanical role of Lpp, the major peptidoglycanassociated lipoprotein and one of the most abundant outer membrane proteins in E. coli cells, has been quantified. We expect that our findings will be helpful in increasing the understanding of the structureproperty relationships of bacterial cell envelopes.The surface layers that isolate the interior of a bacterial cell from its external environment play a crucial mechanical role in the survival of the cell. They must be strong enough to maintain the cellular shape and resist turgor pressure yet, at the same time, be flexible enough to allow cell growth and division. Their elastic response is evident from their ability to recover from transient deformations, such as those induced by the incorporation of additional surface components (e.g., proteins) in response to changes in environmental conditions and the passage of small molecules across the cell boundary. It is therefore clear that understanding many aspects of cell physiology requires knowledge of the mechanical properties of cells.The mechanical properties of the cell originate from the structural organization of the constituent lipids, sugar polymers, and proteins. Lipid molecules are brought together by their hydrophobic domains to form bilayers (membranes) that also incorporate different types of proteins. Polymeric strands of sugar molecules are typically cross-linked by flexible peptide molecules to form the peptidoglycan layer (27). Sometimes, an additional layer of proteins (S layer) is found on the outermost surface of the cell (7,8,40). Depending on the structural organization of the peptidoglycan and lipid bilayers, bacteria can generally be divided into gram-positive and gram-negative bacteria. In gram-positive cells, there is a relatively thick (20-to 35-nm) peptidoglycan layer that, together with the plasma membrane, sandwiches a viscous compartment called the periplasm (31, 32), whereas the envelope of gram-negative cells is made up of two lipid bilayers, the inner and outer membranes, separated by the periplasm, which contains a thin (3-to 8-nm) peptidoglycan layer (5, 33). In gram-negative bacteria, lipoproteins are associated with both the peptidoglycan layer and either the inner or outer membrane. Here, the "lipo" substituent is inserted into the hydrophobic domain of the membrane and the "protein" portion is linked to the peptidoglycan layer by either covalent or electrostatic bonds...