In Situ Characterization of Differences in the Viscoelastic Response of Individual Gram-Negative and Gram-Positive Bacterial Cells

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 peptidoglycan-associated 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 structure-property 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 (18). Loss or altered expression of lipoproteins has been shown to affect cell shape generation and/or membrane integrity (10, 11, 13, 36, 43, 46), suggesting a possible mechanical role for these peptidoglycan-associated proteins.Although the structure and chemistry of the gram-negative and gram-positive bacterial cell envelopes are well known, information about their mechanical properties has been difficult to elucidate. The simple stretching model used by Isaac and Ware (21) to describe the flexibility of bacterial cells indicated differences in the deformability of bacterial cells. Further advances in the characterization of the mechanical properties of bacterial cells were achieved by using bacterial threads, which are so-called macrofibers obtained from cultures of a cell-separation-suppressed mutant that were investigated by standard fiber-testing techniques (34, 48, 49). The requirement to use filament-forming mutants for this mechanical measurement has restricted the studies to date to the gram-positive bacterium Bacillus subtilis. In these studies, bacterial threads of B. subtilis were shown to be viscoelastic by performing creep experiments, a transient rheological technique in which a known force is applied to the material and the resulting extension (or deformation) is measured over time. The properties measured in these experiments were extrapolated to those of the individual cells, often with tenuous lines of inference. Recently, remarkable advances have been made in applying atomic force microscopy (AFM) to quantify the mechanical properties of individual microbial cells (15, 55). Typically, AFM force-indentation curves, which represent the relationship between a loading force and the depth of the indentation as the tip at the end of the AFM cantilever pushes onto the sample surface, are measured. Quantitative information on the elasticity of the sample is then obtained from the force required to achieve a certain depth of penetration (3, 16, 37, 51). It is only recently that direct creep measurements have become possible at the individual cell level by using AFM (50). In AFM creep experiments, the loading force is maintained at a constant value by controlling the cantilever deflection, while the displacement of the cantilever base generated by the sample response to the applied load is measured as a function of time. The sample creep response can be then analyzed with theoretical mechanical models to provide quantitative information on sample viscoelasticity.In the present study, we used AFM creep experiments to probe and compare for the first time the local viscoelastic properties of individual gram-positive (B. subtilis) and gram-negative (Escherichia coli) bacterial cells. 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 peptidoglycan-associated 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 structure-property relationships of bacterial cell envelopes.