Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica
Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica
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DOI：
10.1016/j.femsle.2004.06.013
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发表时间：
2004-08-01
影响因子：
2.1
通讯作者：
Schweyen, RJ
中科院分区：
文献类型：
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作者：
Froschauer, EM;Kolisek, M;Schweyen, RJ
包埋在细胞或细胞器中的Mg ~（2+）荧光染料mag-fura 2经常被用于真核细胞（主要是哺乳动物细胞）中游离离子Mg ~（2+）浓度的双激发比率测定。在这里，我们报告其成功的应用，以测量游离Mg 2+浓度（[Mg 2 +]（i））在沙门氏菌肠道细胞。当保存在标称无Mg 2+缓冲液中时（静息条件），野生型细胞的[Mg 2 +]（i）已被确定为0.9 mM。（[Mg 2 +]（e））导致[Mg 2 +]（i）的快速增加，在约1.5 mM下在几秒内饱和，其中[Mg-2除以]（i）为20 mM。缺乏Mg 2+转运蛋白CorA、MgtA、MgtB的细胞不能显示这种快速增加。相反，它们的[Mg 2 +]（i）在较长的时间内稳定增加，并在低于野生型细胞的浓度下饱和。当corA在这些突变体细胞中过表达时，Mg 2+摄取率增加了15倍以上。Mg 2+的摄取到corA表达细胞中强烈刺激尼日利亚菌素，这增加了膜电位DeltaPsi的代价DeltapH，并大大减少缬氨霉素，这降低了膜电位DeltaPsi。这些结果揭示了mag-fura 2作为测量静息细菌细胞中的稳态[Mg 2 +]（i）值和确定Mg 2+摄取速率的有用指示剂。他们证实了CorA作为主要的Mg 2+转运蛋白的作用，并揭示了膜电位作为Mg 2+吸收到S中的驱动力。肠细胞(C)2004年，欧洲微生物学会联合会。Elsevier B. V.出版，保留所有权利。
The Mg2+ fluorescent dye mag-fura 2, entrapped in cells or organelles, has frequently been used for dual excitation ratio-metric determinations of free ionic Mg2+ concentrations in eukaryotic, mostly mammalian cells. Here we report its successful application to measure free Mg2+ concentrations ([Mg2+](i)) in Salmonella enterica cells. When kept in nominally Mg2+ free buffer (resting conditions), the [Mg2+](i) of wild-type cells has been determined to be 0.9 mM. An increase in the external Mg-2divided by concentration ([Mg2+](e)) resulted in a rapid increase of [Mg2+](i), saturating within a few seconds at about 1.5 mM with [Mg-2divided by](i) of 20 mM. In contrast, cells lacking the Mg2+ transport proteins CorA, MgtA, MgtB failed to show this rapid increase. Instead, their [Mg2+](i) increased steadily over extended periods of time and saturated at concentrations below those of wild-type cells. Mg2+ uptake rates increased more than 15-fold when corA was overexpressed in these mutant cells. Uptake of Mg2+ into corA expressing cells was strongly stimulated by nigericin, which increased the membrane potential DeltaPsi at the expense of DeltapH, and drastically reduced by valinomycin, which decreased the membrane potential DeltaPsi. These results reveal mag-fura 2 as a useful indicator to measure steady-state [Mg2+](i) values in resting bacterial cells and to determine Mg2+ uptake rates. They confirm the role of CorA as the major Mg2+ transport protein and reveal the membrane potential as driving force for Mg2+ uptake into S. enterica cells. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.