Quantification of membrane fluidity in bacteria using TIR-FCS

Area of Science:

• Microbiology
• Biophysics
• Cell Biology

Background:

• Plasma membrane fluidity is crucial for bacterial cell function, impacting protein activity, respiration, transport, and antibiotic resistance.
• Environmental stresses like temperature changes necessitate tight regulation of membrane fluidity for bacterial adaptation.
• Existing methods for measuring membrane fluidity are often unsuitable for small, curved bacterial cells.

Purpose of the Study:

• To develop and validate a novel assay for directly measuring membrane fluidity in live bacteria.
• To quantify bacterial membrane fluidity dynamics in response to temperature variations and cold shock.
• To investigate differences in membrane fluidity regulation between bacterial species.

Main Methods:

• Development of a total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) assay for live bacteria.
• Utilizing simulations validated by experiments to correct for bacterial size, curvature, and geometry in diffusion measurements.
• Quantifying the diffusivity of fluorescent membrane markers to determine membrane fluidity.

Main Results:

• The developed TIR-FCS assay successfully measured bacterial membrane fluidity.
• Steady-state membrane fluidity was lower at 20°C compared to 37°C and varied between Bacillus subtilis and Staphylococcus aureus at 37°C.
• Cold shock caused a further decrease in membrane fluidity, with recovery observed within 30 minutes in both species.

Conclusions:

• The novel TIR-FCS assay provides a minimally invasive tool for studying bacterial membrane fluidity.
• This assay enables quantitative analysis of bacterial adaptation to environmental temperature changes.
• The findings offer new perspectives for studying bacterial membrane responses to various external factors, including antibiotics and viral infections.