Determining the Young's Modulus of the Bacterial Cell Envelope.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of Escherichia coli range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of E. coli and of the pathogens Vibrio cholerae and Staphylococcus aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for E. coli, 0.84 ± 0.02 MPa for E. coli treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for V. cholerae, and 1.52 ± 0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway.

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, 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 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, direct compression and hydrostatic pressure. In all 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. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Cell biomechanics and mechanobiology in bacteria: Challenges and opportunities.

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.

Mechanical stress compromises multicomponent efflux complexes in bacteria.

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 by Escherichia coli to 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.