Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, Δ mscL , Δ mscS , a double knockout Δ mscS Δ mscK , and a triple knockout Δ mscL Δ mscS Δ mscK in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the Δ mscL strain, suggesting either a cross-talk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants. Summary (for the table of contents) The kinetics of hypotonic osmolyte release from E. coli is analyzed in conjunction with bacterial survival. It is shown that MscL, the high-threshold ‘emergency release valve’, rescues bacteria from down-shocks only in the presence of MscS, MscK or other low-threshold channels that are necessary to pacify MscL at the end of the release phase. ### Competing Interest Statement The authors have declared no competing interest.