Measuring the fluidity of the membrane of Bacillus subtilis using TIR-FCS.

Abstract

Bacteria need to constantly maintain the fluidity of their membrane in response to external stimuli such as changes in temperature, growth medium, etc. So far, bacterial membrane remodeling could only be assessed indirectly via biochemical methods like fatty acid analysis or in live bacteria using environment-sensitive dyes. Fluorescence correlation spectroscopy (FCS), a gold-standard technique in membrane dynamics investigations, was however barely used to study bacterial membranes. This is likely due to an increased technical complexity, caused by the small size of bacteria and mounting difficulties, which complicates FCS measurements on confocal microscopes as they are usually performed. Here, we show that imaging FCS on a TIRF microscope (TIR-FCS) can remediate this thanks to an increased axial resolution with respect to confocal and ability to correct for sample drift using image registration. To facilitate the analysis of the large amount of data generated by TIR-FCS, we developed a simple method to automatically exclude artefactual FCS curves. As a result, we could perform high-throughput measurements of the diffusion coefficients of lipophilic dyes like Nile Red or TopFluor-sphingomyelin in the membrane of B. subtilis. Using this assay, we observed the effects of membrane remodeling in response to known stimuli like a cold shock. We also looked at the effects of the widely used membrane-perturbing drug benzyl alcohol, revealing an unexpectedly temperature-dependent fluidization effect. Finally, we used TIR-FCS to explore membrane fluidity adaptation during the sporulation process. We believe that this method has significant potential in microbiology and can easily be applied in the future to different problems, to study membrane proteins diffusion or to understand the effects of selected drugs on membrane viscosity.