Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress

To obtain turgor pressure, intracellular osmolalities, and cytoplasmic wate r activity of Escherichia coli as a function of osmolality of growth, we ha ve quantified and analyzed amounts of cell, cytoplasmic, and periplasmic wa ter as functions of osmolality of growth and osmolality of plasmolysis of n ongrowing cells with NaCl. The effects are large; NaCl (plasmolysis) titrat ions of cells grown in minimal medium at 0.03 Osm reduce cytoplasmic and ce ll water to similar to 20% and similar to 50% of their original values, and increase periplasmic water by similar to 300%. independent analysis of amo unts of cytoplasmic and cell water demonstrate that turgor pressure decreas es with increasing osmolality of growth, from similar to 3.1 atm at 0.03 Os m to similar to 1.5 at 0.1 Osm and to less than 0.5 atm above 0.5 Osm. Anal ysis of periplasmic membrane-derived oligosaccharide (MDO) concentrations a s a function of osmolality, calculated from literature analytical data and measured periplasmic volumes, provides independent evidence that turgor pre ssure decreases with increasing osmolality, and verifies that cytoplasmic a nd periplasmic osmolalities are equal. We propose that MDO play a key role in periplasmic volume regulation at tow-to-moderate osmolality. At high gro wth osmolalities, where only a small amount of cytoplasmic water is observe d, the small turgor pressure of E. coli demonstrates that cytoplasmic water activity is only slightly less than extracellular water activity. From the se findings, we deduce that the activity of cytoplasmic water exceeds its m ole fraction at high osmolality, and, therefore, conclude that the activity coefficient of cytoplasmic water increases with increasing growth osmolali ty and exceeds unity at high osmolality, presumably as a consequence of mac romolecular crowding. These novel findings are significant for thermodynami c analyses of effects of changes in growth osmolality on biopolymer process es in general and osmoregulatory processes in particular in the E. coli cyt oplasm.