Fast Osmotic Permeability Responses in Bacteria

Abstract

Bacteria survive abrupt osmotic downshocks not simply because they are surrounded with a cross-linked peptidoglycan layer, but also because they quickly reduce excessive turgor pressure by releasing ions and small osmolytes. To rescue the cell, the tension-activated (mechanosensitive) channels must ensure that the rate of internal osmolyte release outpaces the osmotic influx of water. Utilizing a stopped-flow apparatus equipped with a small-angle light scattering detector and a conductometry cell, we completed a set of experiments on live E. coli cultures measuring (i) water permeability during up-shocks, (ii) rates of total osmolyte release from light scattering traces and (iii) kinetics of ionic species release from the time course of external conductance increase. Cells shrinking during the up-shock increase the refractive index of their cytoplasm and consequently increase light scattering. In contrast, osmotic downshock leads to a massive release of both ionic and non-ionic osmolytes, decreasing refractive index and increasing external conductivity at the same time. We show the kinetics of osmoadaptation and provide estimated permeabilities for water, ions and total osmolytes in exponential and stationary cultures of WT E. coli and in strains expressing individual channels, MscS or MscL. Interestingly, the increase in conductivity in many cases lags behind the light scattering response, and E. coli pre-loaded with zwitterionic compatible osmolytes such as glycine-betaine exhibit a faster osmolyte release under similar osmotic conditions compared to cells loaded with K+.