| Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R)
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Author:
Date:
2017-09-05
[Abstract] The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum (Minamino, 2014). The export apparatus is a proton/protein antiporter that couples the proton flow with protein transport through the gate complex (Minamino et al., 2011). A transmembrane export gate protein, FlhA, acts as an energy transducer along with the cytoplasmic ATPase complex (Minamino et al., 2016). To directly measure the proton flow through the FlhA channel that is coupled with the flagellar protein export, we have ...
[摘要] 细菌鞭毛III型蛋白质输出装置由跨膜出口门复合物和胞质ATP酶复合物组成。出口设备需要ATP水解和跨细胞质膜的质子动力来展开和转运鞭毛成分蛋白以构建细菌鞭毛（Minamino，2014）。出口设备是质子/蛋白质反向转运体，其将质子流与通过门络合物的蛋白质转运相结合（Minamino等，2011）。跨膜输出门蛋白FlhA与胞质ATP酶复合物一起作为能量转导（Minamino等，2016）。为了直接测量通过与鞭毛蛋白输出相结合的FlhA通道的质子流，我们开发了具有高空间和pH分辨率的体内pH成像系统（Morimoto等，2016）。在这里，我们描述了我们如何测量生活沙门氏菌细胞出口设备附近的局部pH（Morimoto et al。，2016）。我们的方法可以应用于广泛的活细胞。由于局部pH值是监测活细胞活性的最重要参数之一，因此我们的方案将广泛应用于生命科学的各个领域。
【背景】已经检测到跨膜质子通道复合物的质子通道活性是细菌细胞的细胞质pH降低（Morimoto等，2010; Che等，2014; Furukawa等，2017）。然而，为了详细测量活细胞中膜复合物的质子通道活性，需要精确测量局部细胞质pH。绿色荧光蛋白（GFP）的衍生物，pHluorin，激发波长为410和470 nm，发射于508 nm是测量活细胞胞质pH值的有用探针（Miesenböcket ...
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| Bacterial Intracellular Sodium Ion Measurement using CoroNa Green
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Author:
Date:
2017-01-05
[Abstract] The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H+ and Na+ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na+ concentrations in living Escherichia coli ...
[摘要] 细菌鞭毛III型出口设备由细胞质ATP酶复合物和跨膜出口门复合物组成，分别由ATP和质子动力（PMF）驱动跨越细胞质膜，并将鞭毛成分蛋白从细胞质转运到远端结束它们的组装发生的鞭毛结构（Minamino，2014）。当细胞质ATPase复合物不能正常工作时，出口门复合物可以利用除PMF之外的钠动力。跨膜出口门蛋白FlhA充当双离子通道，以进行H + 和Na + （Minamino等人，2016）。在这里，我们描述如何使用钠敏感荧光染料CoroNa Green（Minamino等人）测量活细胞大肠杆菌细胞中的细胞内Na+浓度，。，2016）。通过荧光显微镜检测CoroNa Green的荧光强度，可以定量测定细胞内Na +的浓度。
背景 通过荧光成像技术测量细胞内Na +浓度能够在单细胞水平上更精确和定量地进行，因为每个细胞的背景噪声可以通过图像分析程序去除。 Lo <等等。已经建立了用于测量活体中的细胞质Na +浓度的方案。使用钠敏感荧光染料钠绿，并显示细胞质Na +浓度维持在10mM左右。大肠杆菌在0至100mM的外部Na +浓度范围的宽范围内（Lo et al。，2006）。因为CoroNa ...
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| Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts
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Author:
Date:
2016-10-05
[Abstract] Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) is the first enzyme of the oxidative pentose phosphate cycle and catalyses the conversion of glucose-6-phosphate (G6P) to 6-phosphoglucono-δ-lactone and transfers one electron to NADP+ producing one NADPH. Conversion of G6P to 6-phosphoglucono-δ-lactone is proportional to the production of NADPH. The increase in NADPH concentration results in an increase in absorbance at 340 nm. To assay G6PDH activity, therefore, production of NADPH is determined by measuring increase in absorbance at 340 nm spectrophotometrically. This increase rate is then converted to unit of activity and specific activity of G6PDH. In this procedure, a generalized method is given for bacterial G6PDH assays emphasizing on a cyanobacterium Synechocystis ...
[摘要] 葡萄糖-6-磷酸脱氢酶（G6PDH）（EC 1.1.1.49）是氧化戊糖磷酸循环的第一个酶，并催化葡萄糖-6-磷酸（G6P）转化为6-磷酸葡萄糖酸-δ-内酯并将一个电子 到NADP +产生一种NADPH。 G6P向6-磷酸葡萄糖酸-δ-内酯的转化与NADPH的产生成比例。 NADPH浓度的增加导致340nm处吸光度的增加。 因此，为了测定G6PDH活性，通过分光光度法测量340nm处吸光度的增加来确定NADPH的产生。 然后将该增加速率转化为G6PDH的活性和比活性的单位。 在该程序中，给出了强调蓝细菌集胞藻的细菌G6PDH测定的一般方法。 PCC6803（Schaeffer和Stanier，1978; Karakaya等人，2008，2012）和异养细菌大肠杆菌（Hylemon和Phibbs，1972; Barnel等人 ，1990）。
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