Department of Chemistry, Université du Québec à Montréal, C.P. 8888, Succursale Centre-Ville, Montreal H3C 3P8, Canada.
Department of Chemistry, Université du Québec à Montréal, C.P. 8888, Succursale Centre-Ville, Montreal H3C 3P8, Canada.
Biochim Biophys Acta Biomembr. 2022 Feb 1;1864(2):183819. doi: 10.1016/j.bbamem.2021.183819. Epub 2021 Nov 17.
Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.
细胞标记是多种生物物理方法的初步步骤，包括体内细菌的固态核磁共振（NMR）研究。氘固态 NMR 在过去几年中被用于探测细菌膜及其与抗菌肽的相互作用，遵循标准的标记方案。我们实验室最近对一种生长缓慢的细菌的研究结果表明，需要优化该方案，特别是在收获前细菌的生长时间和用于大肠杆菌和枯草芽孢杆菌的外源性标记脂肪酸的浓度。该方案在为细胞提供最佳标记的同时保持对细胞无害也是至关重要的。因此，我们开发了一种快速简便的方法，结合溶液 P NMR 和 GCMS 来监测各种生长条件下细菌膜的脂质组成。使用这种方法，确定了用氘化棕榈酸对大肠杆菌和枯草芽孢杆菌进行最佳标记的条件。我们的结果表明，枯草芽孢杆菌的磷脂谱随着生长阶段的变化而变化，而大肠杆菌则没有。我们的方案建议在生长培养基中添加低浓度的外源性棕榈酸，并在指数生长期后收获细菌。
