Substrate stiffness modulates bacterial adhesion and diversity of adherent phenotypes across growth stages

Surface-adhesion and stiffness of underlying substrates mediate geometry, mechanics and self-organization of bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment, yet the variation of cell-to-surface adhesion with substrate stiffness remains to be quantified. Here, by developing a cell-level Force Distance Spectroscopy (FDS) technique based on Atomic Force Microscopy (AFM), we simultaneously quantify the cell-surface adhesion alongside stiffness of the underlying substrates to reveal stiffness-dependent adhesion in phototrophic bacterium Chromatium okenii. As stiffness of the soft substrate, modelled via low-melting-point (LMP) agarose pad, was varied between 20 kPa and 120 kPa by changing agarose concentrations, we observe a progressive increase of the mean adhesion force by over an order of magnitude, from 0.21 (+/-0.10) nN to 2.42 (+/-1.16) nN. In contrast, passive polystyrene (PS) microparticles of comparable dimensions showed no perceptible change in their surface adhesion. Furthermore, for Escherichia coli, the cell-surface adhesion varied between 0.29 (+/-0.17) nN to 0.39 (+/-0.20) nN, showing a weak dependence on the substrate stiffness, thus suggesting that the stiffness-modulated adhesion is a species-specific trait. Finally, by quantifying the adhesion of C. okenii populations across growth stages, we report an emergent co-existence of weak and strongly adherent sub-populations, demonstrating a diversification of adherent phenotypes over time. Taken together, these findings suggest that bacteria, depending on the species and their physiological stage, actively modulate cell-to-surface adhesion in response to substrate stiffness, and leverage it as a functional trait to modulate initial attachment and colonization on soft substrates during early stages of biofilm development.