Measurement of the Viscosity of E. coli Membranes using Molecular Rotors and Flim

We have employed molecular rotors, small organic molecules whose fluorescence lifetime is sensitive to the viscosity of the environment, to assess the viscosity of the E.coli plasma membrane. We used Fluorescence Lifetime Imaging Microscopy (FLIM) which allowed us to measure the fluorescence lifetimes (and thus viscosities) on the level of single cells. We probed the viscosity of membranes both in livee cells and in spheroplasts, where the outer membrane was removed by lysozyme treatment. Viscosity values obtained for both environments were similar implying that the molecular rotor used indeed localized to the plasma membrane as shown previously for fluorophores of similar structures. Measurements on life cells show a rather broad spread of viscosities between individual cells in population; such heterogeneity of physical parameters of the cell has been reported previously for the diffusion of protein in the cytoplasm of bacteria. The viscosity of membranes was temperature dependent as we have observed a change in viscosity when cells grown at 37 degrees Celsius were measured at lower temperatures than the growth. Subjecting the cells to a hyperosmotic shock by increasing the medium osmolarity by adding NaCl also elicited a change in viscosity and yet a larger spread of viscosity values between individual cells, which is consistent with previous observations that a fraction of cell within a population seems to respond to the osmotic shock more strongly than the others. The values of viscosity measured for the plasma membrane of E.coli in this study are higher than those measured previously in E.coli lipid extracts or in the plasma membrane of life eukaryotic cells but slightly lower than what was reported previously for the Gram-positive bacterium Bacillus subtilis.