A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester
A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester
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DOI：
10.1128/aem.62.1.178-183.1996
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发表时间：
1996-01-01
影响因子：
4.4
通讯作者：
Abee, T
中科院分区：
文献类型：
--
作者：
Breeuwer, P;Drocourt, JL;Abee, T
建立了一种基于荧光探针5（和6-）-羧基荧光素琥珀酰亚基酯（cFSE）细胞内偶联的新方法来测定细菌的细胞内pH， cFSE可以被细菌以其二乙酸酯的形式吸收，5（和6-）-羧基荧光素琥珀酰亚基酯随后在细胞质中被酯酶水解为cFSE，当乳酸乳球菌细胞被乙醇渗透时，cFSE在细胞中保留了很大比例，这表明cFSE是结合在细胞内的，在可发酵糖的存在下，通过细胞短暂的孵育可以方便地挤出未结合的探针，很可能是利用了一个主动的运输系统，这种cFSE的运输系统在乳酸乳杆菌、innocuia李斯特菌和枯草芽孢杆菌中被发现。细菌胞内pH值可通过pa敏感波长（490 nm）荧光信号与pH不敏感波长（440 nm）荧光信号的比值测定。这种cFSE比值法显著减少了在测量过程中由于荧光探针从细胞中流出而引起的问题。此外，所描述的方法已成功地用于确定细菌在压力条件下的细胞内pH值，如高温和洗涤剂的存在。
A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria, cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm, When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly, Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system, Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis, The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pa-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.