The Response of Single E. Coli Cells to Changes in External Osmolarity

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The extreme concentrations of chemicals in a bacterium's cytoplasm generate a large osmotic pressure that inflates the cell. Using a number of interconnected systems, bacteria actively regulate their turgor pressure to resist changes in their local environment. In response to osmotic shock and changes in internal ion concentration, the osmosensory transporters ProP/U, BetT/U, TrkAH and KdpFABC transport external chemicals such as proline, choline and potassium into the cell, whereas the mechano-sensitive channels MscS and MscL export solutes from the cell in response to increased membrane strain. Although each has been shown to play a role in the regulation of turgor, details of how the different systems are coordinated by a cell is poorly understood. Previous measurements of osmoregulation in bacteria have been unable to directly probe the adaptation of turgor pressure, focusing instead on the activity of various transporters, or the change in cellular survival rates. Here we move beyond these limited measurements using AFM and fluorescence imaging to monitor turgor pressure and cell volume adaptation on a single cell level with a time resolution on the order of seconds. To explore different mechanisms used by bacteria, we moved exponentially growing cells from LB medium to an iso-osmotic buffered medium and allowed the cells to adapt. We subsequently challenged the cells with varying levels of sucrose (as an external osmolyte) and potassium or proline. We measured the dependence of the adaptation time and adaptation level on different amounts of extracellular potassium or proline and the magnitude of the osmotic shock. This type of measurement allows us to uncouple the different adaptation pathways and to study them individually and in small groups to quantify their function and interactions.