Surface structure and nanomechanical properties of Shewanella putrefaciens bacteria at two pH values (4 and 10) determined by atomic force microscopy

Abstract

The nanomechanical properties of gram-negative bacteria (Shewanella putrefaciens) were investigated in situ in aqueous solutions at two pH values, specifically, 4 and 10, by atomic force microscopy (AFM). For both pH values, the approach force curves exhibited subsequent nonlinear and linear regimens that were related to the progressive indentation of the AFM tip in the bacterial cell wall, including a priori polymeric fringe (nonlinear part), while the linear part was ascribed to compression of the plasma membrane. These results indicate the dynamic of surface ultrastructure in response to changes in pH, leading to variations in nanomechanical properties, such as the Young's modulus and the bacterial spring constant.

FIG. 1.

Means and respective standard deviations of force curves at different pHs measured on the top of bacteria in aqueous electrolyte solution (KNO₃ = 0.1 M). The two dashed lines indicate linear behaviors that corresponded using equation 1 to bacterial spring constants of 0.05 N/m (pH 4) and 0.02 N/m (pH 10). The onset of the linear regimen corresponds to a loading force of 0.58 nN at a relative piezo displacement of −120 nm (−62 nm without cantilever deflection) for pH 4 and 0.54 nN at a distance of −190 nm (−136 nm without cantilever deflection) for pH 10. The zero piezo displacement was positioned to the lift-off from zero deflection on the approach curve.

FIG. 2.

Mean nano-identation curves obtained under the two pH conditions, pH 4 (a) and pH 10 (b), for S. putrefacians cells at 0.1 M KNO₃. The curves were fitted by summing the contributions of the Hertz model (dashed line) and linear behavior with the bacterial spring constant (dotted line) to reproduce the complete indentation curves as the solid line (equation 2). Best fitting was achieved for an E of 0.21 MPa and a k_B of 0.022 N/m (a) and for an E of 0.037 MPa and a k_B of 0.01 N/m (b). The onset of the linear regimen (dotted line) corresponds to a loading force of 0.48 nN at an indentation of 60 nm for pH 4 and of 0.53 nN at an indentation of 155 nm for pH 10.

FIG. 3.

Schematic view of the dynamic of the cell envelope in response to a change in pH from 4 to 10 (not drawn to scale). OM, outer membrane; IM, inner membrane.