Direct Measurement of Cell Wall Stress Stiffening and Turgor Pressure in Live Bacterial Cells

Abstract

We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of 1.22 ± 0.12, such that the wall is significantly stiffer in intact cells (E = 23 ± 8 MPa and 49 ± 20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29 ± 3 kPa.Many cellular-scale processes in biology, such as cell growth, division and motility, necessarily involve mechanical interactions. Recent theoretical work in bacteria has led to a number of physically-realistic models of bacterial cells [1][2][3]. However, in many instances, precise, direct measurements of the mechanical properties of cellular components in live cells are lacking.The cell envelope in most bacteria is made of one or two layers of membrane and a rigid cell wall consisting of a network of peptidoglycan (PG) polymers. These two materials serve different cellular functions. The semipermeable plasma membrane maintains a chemical separation between the cell interior and the surrounding medium. The large concentration of solutes in the cytoplasm generates an osmotic pressure, termed turgor pressure, that pushes the plasma membrane against the cell wall. The cell wall, on the other hand, defines the cell shape and constrains the volume under turgor.The magnitude of the turgor pressure under physiological conditions has been estimated using several techniques: by collapsing gas vesicles in rare species of bacteria [4], by AFM indentation [5, 6], and by calculating the total chemical content of the cytoplasm [7]. The estimated pressure values vary by more than an order of magnitude, from 10 4 to 3 × 10 5 Pa. While mechanical experiments, such as AFM indentation, are the most direct probes, separating the mechanical contributions of the wall and pressure has not been previously possible and thus these experiments may only provide an upper bound on the true turgor pressure.Similarly, the elasticity of the cell wall has been difficult to probe in individual, live cells. Most previous mechanical measurements on the cell wall have been performed using chemically isolated walls, termed sacculi, that may be altered from the native state, or on large bundles of cells [8]. Yao et al. reported an anisotropic elasticity of 25 MPa and 45 MPa in the axial and circumferential directions relative to a cell's rod-shape using single flattened E. coli sacculi adhered to a substrate [9]. Thwaites and coauthors probed the elastic modulus of macroscopic threads of many Bacillus subtilis sacculi in humid air and found that the modulus varied from 10 to 30 MPa depending on the humidity and salt concentration [10][11][12]. Attempts to probe whole-cell elasticity have also been made using AFM indentation of Myxococcus xanthus cells [13] and optical-tweezer bending of Borrelia burgdorf...

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