For most cells, a sudden decrease in external osmolarity results in fast water influx that can burst the cell. To survive, cells rely on the passive response of mechanosensitive channels, which open under increased membrane tension and allow the release of cytoplasmic solutes and water. Although the gating and the molecular structure of mechanosensitive channels found in Escherichia coli have been extensively studied, the overall dynamics of the whole cellular response remain poorly understood. Here, we characterize E. coli's passive response to a sudden hypoosmotic shock (downshock) on a single-cell level. We show that initial fast volume expansion is followed by a slow volume recovery that can end below the initial value. Similar response patterns were observed at downshocks of a wide range of magnitudes. Although wild-type cells adapted to osmotic downshocks and resumed growing, cells of a double-mutant (ΔmscL, ΔmscS) strain expanded, but failed to fully recover, often lysing or not resuming growth at high osmotic downshocks. We propose a theoretical model to explain our observations by simulating mechanosensitive channels opening, and subsequent solute efflux and water flux. The model illustrates how solute efflux, driven by mechanical pressure and solute chemical potential, competes with water influx to reduce cellular osmotic pressure and allow volume recovery. Our work highlights the vital role of mechanosensation in bacterial survival.osmotic downshock | bacterial mechanosensing | single-cell imaging B iology offers an array of intriguing mechanical solutions, both active and passive, often exceeding what is currently possible with man-made methods. Understanding how biological systems achieve different functionalities under mechanical stimuli can inform new, thus-far-unexplored design principles. One such passive control system is the bacterial response to sudden decreases in external osmolarities.A Gram-negative cell's fluid cytoplasm is separated from the external environment by the inner membrane, the periplasmic space, and the outer membrane. Ordinarily, the total solute concentration within the cytoplasm is higher than that of the environment, resulting in a positive osmotic pressure on the cell wall (termed turgor pressure) (1). Escherichia coli is able to respond to both increases and decreases in external concentrations. An increase in external osmolarity (hyperosmotic shock or upshock) results in water efflux from the cell interior, causing cellular volume to shrink and osmotic pressure to drop to zero (2). E. coli responds by actively accumulating specific solutes (osmolytes), such as potassium, proline, and glycine-betaine (2). Accumulation of osmolytes in the cell's cytoplasm causes reentry of water, cell volume increase, and recovery of osmotic pressure (3, 4). A downward shift in external osmolarity (termed hypoosmotic shock or downshock) causes fast water influx into the cell's cytoplasm. As a result, the osmotic pressure increases and the cell expands in a nonlinear fashion (5,6

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Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to β-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating β-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features—turgor pressure, mechanosensitive channels, and cell shape changes—that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to β-lactams aimed at targeting cellular turgor.

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Experiments have suggested that bacterial mechanosensitive channels separate into 2D clusters, the role of which is unclear. By developing a coarse-grained computer model we find that clustering promotes the channel closure, which is highly dependent on the channel concentration and membrane stress. This behaviour yields a tightly regulated gating system, whereby at high tensions channels gate individually, and at lower tensions the channels spontaneously aggregate and inactivate. We implement this positive feedback into the model for cell volume regulation, and find that the channel clustering protects the cell against excessive loss of cytoplasmic content.