Quantification of membrane fluidity in bacteria using TIR-FCS.

Plasma membrane fluidity is an important phenotypic feature that regulates the diffusion, function and folding of transmembrane and membrane-associated proteins. In bacterial cells, variations in membrane fluidity are known to affect respiration, transport and antibiotic resistance. Membrane fluidity must therefore be tightly regulated to adapt to environmental variations and stresses like temperature fluctuations or osmotic shocks. Quantitative investigation of bacterial membrane fluidity has been however limited due to the lack of available tools, primarily due to the small size and membrane curvature of bacteria that precludes most conventional analysis methods used in eukaryotes. Here, we developed an assay based on total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. With simulations validated by experiments, we could determine how the small size, high curvature and geometry of bacteria affects diffusion measurements and correct subsequent measurements for unbiased diffusion coefficients estimation. We used this assay to quantify the fluidity of the cytoplasmic membranes of the Gram-positive bacteria Bacillus subtilis (rod-shaped) and Staphylococcus aureus (coccus) at high (37°C) and low (20°C) temperatures in a steady-state and in response to a cold shock, caused by a shift from high to low temperature. The steady-state fluidity was lower at 20°C than at 37°C, yet differed between B. subtilis and S. aureus at 37°C. Upon cold shock, the membrane fluidity decreased further below the steady-state fluidity at 20°C and recovered within 30 mins in both bacterial species. Our minimally invasive assay opens up exciting perspectives for the study of a wide range of phenomena affecting the bacterial membrane, from disruption by chemicals or antibiotics to viral infection or change in nutrient availability.

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