Oxygen isotopes indicate most intracellular water in log-phase Escherichia coli is derived from metabolism.

The transport of water into and out of unicellular organisms is a seemingly simple process in which water diffuses either through pores or directly across the membrane. Because of the rates at which water can theoretically diffuse, it is generally accepted that intracellular water is indistinguishable from extracellular water. Using stable isotope ratio mass spectrometry, we directly tested this assumption. Here we demonstrate that under active growth, up to 70% of the intracellular water in log-phase Escherichia coli cells was actually generated during metabolism and was isotopically distinct from extracellular water. The contribution of metabolism to intracellular water was substantially less in stationary-phase or quiescent cells.