Non-invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator
Non-invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator
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DOI：
10.1111/1751-7915.13439
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发表时间：
2019-07-01
影响因子：
5.7
通讯作者：
Nikel, Pablo I.
中科院分区：
文献类型：
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作者：
Arce-Rodriguez, Alejandro;Volke, Daniel C.;Nikel, Pablo I.
假单胞菌在所有主要自然环境（即陆地，淡水和海洋）中茁壮成长的能力是基于其适应物理化学变化的特殊能力。因此，环境细菌必须在不同条件下严格控制许多生理特性的维持。细胞内pH（pH（i））稳态是一个特别重要的特征，因为pH（i）影响细胞中大部分的生化过程。尽管其重要性，相对较少的可靠的，易于实施的工具已被设计用于定量在革兰氏阴性菌的体内pH（i）的变化，以最小的操作。在这里，我们描述了一种方便的，非侵入性的协议，用于定量的pH值（i）在细菌中，这是基于比率荧光指示蛋白PHP（假单胞菌的pH指示剂）。对编码PHP的DNA序列进行了彻底调整，以保证指示剂在假单胞菌属物种中的最佳转录和翻译。我们的基于PHP的定量方法表明，pH（i）不仅在假单胞菌中，而且在其他革兰氏阴性细菌物种（如大肠杆菌）中，在一个狭窄的pH值范围内受到严格调节。在内部（例如恶臭假单胞菌中葡萄糖消耗途径的重定向）和外部（例如铜绿假单胞菌中抗生素暴露）扰动时也可以观察到体内细胞质pH稳态的维持，并且PHP指示剂也用于跟踪外部pH变化时pH（i）的动态变化。总之，我们的工作描述了一个可靠的方法来测量pH值（i）在假单胞菌，允许细菌pH值（i）稳态及其调节的详细调查。
The ability of Pseudomonas species to thrive in all major natural environments (i.e. terrestrial, freshwater and marine) is based on its exceptional capability to adapt to physicochemical changes. Thus, environmental bacteria have to tightly control the maintenance of numerous physiological traits across different conditions. The intracellular pH (pH(i)) homoeostasis is a particularly important feature, since the pH(i) influences a large portion of the biochemical processes in the cell. Despite its importance, relatively few reliable, easy-to-implement tools have been designed for quantifying in vivo pH(i) changes in Gram-negative bacteria with minimal manipulations. Here we describe a convenient, non-invasive protocol for the quantification of the pH(i) in bacteria, which is based on the ratiometric fluorescent indicator protein PHP (pH indicator for Pseudomonas). The DNA sequence encoding PHP was thoroughly adapted to guarantee optimal transcription and translation of the indicator in Pseudomonas species. Our PHP-based quantification method demonstrated that pH(i) is tightly regulated over a narrow range of pH values not only in Pseudomonas, but also in other Gram-negative bacterial species such as Escherichia coli. The maintenance of the cytoplasmic pH homoeostasis in vivo could also be observed upon internal (e.g. redirection of glucose consumption pathways in P. putida) and external (e.g. antibiotic exposure in P. aeruginosa) perturbations, and the PHP indicator was also used to follow dynamic changes in the pH(i) upon external pH shifts. In summary, our work describes a reliable method for measuring pH(i) in Pseudomonas, allowing for the detailed investigation of bacterial pH(i) homoeostasis and its regulation.