Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell wall and therefore it is critical for growth. Since changes in extracellular osmolarity have the same physicochemical effects on cells from all biological kingdoms, the response to osmotic stress may be similar in all organisms. Generally, (micro)organisms respond to hyperosmotic stress by rapidly accumulating compatible solutes to prevent the loss of water and loss of turgor pressure. Upon hypo-osmotic stress, the cells need to rapidly exit the solutes to prevent the turgor pressure to become too high, which, ultimately, may lead to breakage of cells. The research in this project is aimed at the elucidation of the osmosensing and regulatory mechanisms that allow cells to respond to hyper- and hypo-osmotic conditions. In Lactococcus lactis an osmoregulated ABC-transporter (OpuA) with novel structural features has been identified that responds to hyperosmotic stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit and a protein that contains both the translocator and the substrate binding domain. Genome analysis has revealed that this novel-type of ABC transporter is present in a wide variety of species, including Helicobacter pylori, Enterococcus faecalis, and others. The osmoregulated OpuA system has been overexpressed, purified, and functionally incorporated into liposomes. A trans-membrane osmotic gradient of both ionic (salts) and non-ionic compounds (sugars) is able to osmotically activate OpuA in the proteoliposomal system. This excludes the possibility that additional cellular factors are involved in the regulation of the transporter, and shows that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic amphipaths, which interact with the membrane. This suggests that the activation by a trans-membrane osmotic gradient is mediated by changes in membrane properties and protein-lipid interactions. The mechanism of the osmotic activation of OpuA has been studied in proteoliposomes of different lipid composition, and analysis of (proteo)liposome properties/structure by spectroscopic techniques, isothermal titration calorimetry, and cryo-electron microscopy (in collaboration with A.D.R. Brisson and M. Stuart). The results indicate that changes in the headgroup region, in particular the molar fraction of anionic lipids, determine the threshold value for osmotic activation.