Two temperature-dependent membrane fluidity regimes in gram-positive bacteria

Temperature changes directly affect the mobility of molecules within a biological membrane, increasing or decreasing its fluidity. Plasma membrane fluidity is a fundamental physical property that governs bacterial cell physiology, influencing processes such as protein function, respiration, and antibiotic uptake. It is widely accepted that bacteria actively maintain a constant membrane fluidity in response to temperature fluctuations to preserve membrane function. This process, known as membrane fluidity homeostasis, occurs through regulated remodeling of lipid composition. Here, we used an assay based on total internal reflection-fluorescence correlation spectroscopy to directly quantify membrane fluidity in the gram-positive model bacterium Bacillus subtilis and two gram-positive pathogens, Streptococcus pneumoniae and Staphylococcus aureus , across a temperature range of 20°C to 37°C. Instead of the expected constant membrane fluidity, we revealed a two-component regime: membrane fluidity is maintained at low temperatures (<26°C) but freely increases at higher temperatures. Far from contradicting previous evidence about thermoadaptation in bacteria, and especially in B. subtilis , our results demonstrate that their usual interpretation is incorrect and provide a framework for studying membrane fluidity across bacterial species. IMPORTANCE Temperature changes affect the physical properties of the plasma membrane. Typically, a reduction in temperature causes a less fluid and thus more viscous, membrane. It has been long established that bacteria such as the model bacterium Bacillus subtilis adapt their membrane composition to maintain membrane fluidity constant under changing temperatures, to survive environmental changes. By directly quantifying membrane fluidity across temperatures in B. subtilis and two other gram-positive bacteria, we found that this long-standing view is not true: fluidity is maintained only at low temperatures (<26°C), while at higher temperatures, it increases linearly with temperature. Our findings present a conceptual advance that broadens our understanding of thermoadaptation and refines the current model of membrane fluidity homeostasis in bacteria.