Poster De Conférence Année : 2023

Measuring membrane fluidity in Bacillus subtilis using TIR-FCS

Résumé

Bacterial cells need to constantly maintain the fluidity of their cytoplasmic membrane in response to external stimuli such as changes in temperature, growth medium, etc. Typically, in response to a cold shock, B. subtilis increases branching and unsaturation of its membrane fatty acids to increase membrane fluidity. So far, bacterial membrane fluidity was mostly assessed indirectly, via analytical methods such as fatty acid analysis or in live bacteria using environment-sensitive dyes. These dyes, like Laurdan, insert in the cytoplasmic membrane and change their fluorescence emission properties when the local environment changes, informing about the local membrane fluidity. Fluorescence correlation spectroscopy (FCS), a gold-standard technique in membrane dynamics investigations in eukaryotic systems, has however been barely used to study bacterial membranes, most likely due to increased technical complexity due to the small size of bacterial cells. Here, we developed an assay using FCS on a total internal reflexion microscope (TIR-FCS) to measure membrane fluidity in bacterial cells. Using TIR-FCS, we could measure the diffusion speed of the membrane marker Nile Red in the membrane of the model Gram-positive bacterium B. subtilis, and link relative changes of its diffusion speed to changes in membrane fluidity. As a proof of concept, we used this technique to observe the effects of membrane remodelling in response to a cold shock in B. subtilis. We also deployed TIR-FCS to monitor the membrane adaptation of the mother cell, which isolates the spore development from its environment, at late stages of sporulation. We believe that our TIR-FCS methodology has significant potential in microbiology and can be easily applied in the future to investigate a wide range of molecular dynamics and physiological adaptations in bacteria.

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Dates et versions

hal-04300629 , version 1 (22-11-2023)

Identifiants

  • HAL Id : hal-04300629 , version 1

Citer

Aurélien Barbotin, Armand Lablaine, Cyrille Billaudeau, Erdinc Sezgin, Rut Carballido-López. Measuring membrane fluidity in Bacillus subtilis using TIR-FCS. New approaches and concepts in microbiology, Jun 2023, Heidelberg (Germany), Germany. ⟨hal-04300629⟩
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