Changes in the extracellular osmolarity can be a fatal event for the bacterial cell. Upon a hypo-osmotic shock, water rushes into the cell across the membrane, leaving the cell with no choice but to equalize the pressure. This equalization occurs either through damage to the cell membrane (resulting in death) or through the regulated flux of water molecules through transmembrane protein channels (Fig 1A). Such proteinaceous pressure release valves have been found across all domains of life, with the first bacterial channel being described in 1987 (1). Over the past thirty years, several more channels have been discovered, described, and (in many cases) biophysically characterized. E. coli, for example, has seven of these channels (one MscL and six MscS homologs) which have varied conductance, gating mechanisms, and expression levels. While they have been the subject of much experimental and theoretical dissection, much remains a mystery with regard to the roles their abundance and interaction with other cellular processes play in the greater context of physiology (2–8).

      Of the seven channels in E. coli, the mechanosensitive channel of large conductance (MscL) is one of the most abundant and the best characterized. This channel has a large conductance (3 nS) and mediates the flux of water molecules across the membrane via a ~3 nm wide pore in the open state (9, 10). Molecular dynamics simulations indicate that a single open MscL channel permits the flux of \(4 \times 10^9\) water molecules per second, which is an order of magnitude larger than a single aquaporin channel (BNID 100479) (11, 12). This suggests that having only a few channels per cell could be sufficient to relieve even large changes in membrane tension. Electrophysiological experiments have suggested a small number of channels per cell (13, 14), however, more recent approaches using quantitative western blotting, fluorescence microscopy, and proteomics have measured several hundred MscL per cell (3, 15, 16). To further complicate matters, the expression profile of MscL appears to depend on growth phase, available carbon source, and other environmental challenges (3, 16, 17). While there are likely more than just a few channels per cell, why cells seem to need so many and the biological rationale behind their condition-dependent expression both remain a mystery.

      While their biochemical and biophysical characteristics have received much attention, their connection to cell survival is understudied. Drawing such a direct connection between channel copy number and survival requires quantitative in vivo experiments. To our knowledge, the work presented in van den Berg et al. 2016 (8) is the first attempt to simultaneously measure channel abundance and survivability for a single species of mechanosensitive channel. While the measurement of channel copy number was performed at the level of single cells using super-resolution microscopy, survivability after a hypo-osmotic shock was assessed in bulk plating assays which rely on serial dilutions of a shocked culture followed by counting the number of resulting colonies after incubation. Such bulk assays have long been the standard for querying cell viability after an osmotic challenge. While they have been highly informative, they reflect only the mean survival rate of the population, obfuscating the variability in survival of the population. The stochastic nature of gene expression results in a noisy distribution of MscL channels rather than a single value, meaning those found in the long tails of the distribution have quite different survival rates than the mean but are lost in the final calculation of survival probability.

       In this work, we present an experimental system to quantitatively probe the interplay between MscL copy number and survival at single-cell resolution, as is seen in Fig. 1B. We generated an E. coli strain in which all seven mechanosensitive channels had been deleted from the chromosome followed by a chromosomal integration of a single gene encoding an MscL-super-folder GFP (sfGFP) fusion protein. To explore copy number regimes beyond those of the wild-type expression level, we modified the Shine-Dalgarno sequence of this integrated construct allowing us to cover nearly three decades of MscL copy number. To probe survivability, we exposed cells to a large hypo-osmotic shock at controlled rates in a flow cell under a microscope, allowing the observation of the single-cell channel copy number and the resulting survivability of single cells. With this large set of single cell measurements, we approach the calculation of survival probability in a manner that is free of binning bias which allows the reasonable extrapolation of survival probability to copy numbers outside of the observed range. In addition, we show that several hundred channels are needed to convey high rates of survival and observe a minimum number of channels needed to permit any degree of survival.