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.
Physical forces play a profound role in the survival and function of all known forms of life. Advances in cell biomechanics and mechanobiology have provided key insights into the physiology of eukaryotic organisms, but much less is known about the roles of physical forces in bacterial physiology. This review is an introduction to bacterial mechanics intended for persons familiar with cells and biomechanics in mammalian cells. Bacteria play a major role in human health, either as pathogens or as beneficial commensal organisms within the microbiome. Although bacteria have long been known to be sensitive to their mechanical environment, understanding the effects of physical forces on bacterial physiology has been limited by their small size (∼1 μm). However, advancements in micro- and nano-scale technologies over the past few years have increasingly made it possible to rigorously examine the mechanical stress and strain within individual bacteria. Here, we review the methods currently used to examine bacteria from a mechanical perspective, including the subcellular structures in bacteria and how they differ from those in mammalian cells, as well as micro- and nanomechanical approaches to studying bacteria, and studies showing the effects of physical forces on bacterial physiology. Recent findings indicate a large range in mechanical properties of bacteria and show that physical forces can have a profound effect on bacterial survival, growth, biofilm formation, and resistance to toxins and antibiotics. Advances in the field of bacterial biomechanics have the potential to lead to novel antibacterial strategies, biotechnology approaches, and applications in synthetic biology.
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
In tissues with mechanical function, the regulation of remodeling and repair processes is often controlled by mechanosensitive mechanisms; damage to the tissue structure is detected by changes in mechanical stress and strain, stimulating matrix synthesis and repair. While this mechanoregulatory feedback process is well recognized in animals and plants, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress and strain within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, compression, and hydrostatic pressure. In all three cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Mechanosensitivity of VxrAB signaling was lost following removal of the VxrAB stimulating endopeptidase ShyA, suggesting that VxrAB may not be directly sensing mechanical forces, but instead relies on other factors including lytic enzymes in the periplasmic space. Our findings suggest that mechanical signals play an important role in regulating cell wall homeostasis in bacteria.Significance StatementBiological materials with mechanical function (bones, muscle, etc.) are often maintained through mechanosensitive mechanisms, in which damage-induced reductions in stiffness stimulate remodeling and repair processes that restore mechanical function. Here we show that a similar process can occur in bacteria. We find that mechanical stresses in the bacterial cell envelope (the primary load-bearing structure in bacteria) regulate signaling of a two-component system involved in cell wall synthesis. These findings suggest that the mechanical stress state within the cell envelope can contribute to cell wall homeostasis. Furthermore, these findings demonstrate the potential to use mechanical stimuli to regulate gene expression in bacteria.
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