Physical forces have a profound effect on growth, morphology, locomotion, and survival of organisms. At the level of individual cells, the role of mechanical forces is well recognized in eukaryotic physiology, but much less is known about prokaryotic organisms. Recent findings suggest an effect of physical forces on bacterial shape, cell division, motility, virulence, and biofilm initiation, but it remains unclear how mechanical forces applied to a bacterium are translated at the molecular level. In Gram-negative bacteria, multicomponent protein complexes can form rigid links across the cell envelope and are therefore subject to physical forces experienced by the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that octahedral shear (but not hydrostatic) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used byEscherichia colito resist copper and silver toxicity. By promoting disassembly of this protein complex, mechanical forces within the cell envelope make the bacteria more susceptible to metal toxicity. These findings demonstrate that mechanical forces can inhibit the function of cell envelope protein assemblies in bacteria and suggest the possibility that other multicomponent, transenvelope efflux complexes may be sensitive to mechanical forces including complexes involved in antibiotic resistance, cell division, and translocation of outer membrane components. By modulating the function of proteins within the cell envelope, mechanical stress has the potential to regulate multiple processes required for bacterial survival and growth.

19 20 21 22 Physical forces have long been recognized for their effects on the growth, 23 morphology, locomotion, and survival of eukaryotic organisms 1 . Recently, mechanical 24 forces have been shown to regulate processes in bacteria, including cell division 2 , motility 3 , 25 virulence 4 , biofilm initiation 5,6 , and cell shape 7,8 , although it remains unclear how 26 mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-27 negative bacteria, multicomponent protein complexes that form rigid links across the cell 28 envelope directly experience physical forces and mechanical stresses applied to the cell. 29Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and 30 use single-molecule tracking to show that shear (but not tensile) stress within the cell 31 envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by 32 E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to 33 metal toxicity. These findings provide the first demonstration that mechanical forces, such 34 as those generated during colony overcrowding or bacterial motility through submicron 35 pores, can inhibit the contact and function of multicomponent complexes in bacteria. As 36 multicomponent, trans-envelope efflux complexes in bacteria are involved in many 37 processes including antibiotic resistance 9 , cell division 10 , and translocation of outer 38 membrane components 11 , our findings suggest that the mechanical environment may 39 regulate multiple processes required for bacterial growth and survival. 40

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement.