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

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