Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France.
Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France.
Biophys J. 2024 Aug 20;123(16):2484-2495. doi: 10.1016/j.bpj.2024.06.012. Epub 2024 Jun 13.
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 such as 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 preclude most conventional analysis methods used in eukaryotes. Here, we develop 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 affect diffusion measurements and correct subsequent measurements for unbiased diffusion coefficient 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 min 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.
质膜流动性是一种重要的表型特征，它调节着跨膜和膜相关蛋白的扩散、功能和折叠。在细菌细胞中，膜流动性的变化已知会影响呼吸、运输和抗生素耐药性。因此，膜流动性必须受到严格的调节，以适应环境变化和压力，如温度波动或渗透压冲击。然而，由于缺乏可用的工具，细菌膜流动性的定量研究受到了限制，主要是由于细菌的体积小和膜曲率大，排除了大多数在真核生物中使用的常规分析方法。在这里，我们开发了一种基于全内反射-荧光相关光谱（TIR-FCS）的测定方法，通过荧光膜标记物的扩散来直接测量活细菌中的膜流动性。通过实验验证的模拟，我们可以确定细菌的小尺寸、高曲率和几何形状如何影响扩散测量，并对后续测量进行校正，以进行无偏扩散系数估计。我们使用该测定法在稳态下和响应冷休克（由从高温到低温的转变引起）时，在高温（37°C）和低温（20°C）下定量测定革兰氏阳性菌枯草芽孢杆菌（杆状）和金黄色葡萄球菌（球菌）的细胞质膜的流动性。在 20°C 时的稳态流动性比 37°C 时低，但在 37°C 时，枯草芽孢杆菌和金黄色葡萄球菌之间存在差异。在冷休克时，膜流动性进一步低于 20°C 时的稳态流动性，并在两种细菌中都在 30 分钟内恢复。我们的微创测定法为研究影响细菌膜的一系列现象开辟了令人兴奋的前景，这些现象包括化学物质或抗生素的破坏、病毒感染或营养物质可用性的变化。
