Monitoring the intracellular pH of Zygosaccharomyces bailii by green fluorescent protein

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• 作者：T.D.T. Dang^a ; S.L. De Maeseneire^b ; B.Y. Zhang^a ; W.H. De Vos^c ; A. Rajkovic^a ; ^d ; A. Vermeulen^a ; J.F. Van Impe^e ; F. Devlieghere^a ; ^{Frank.Devlieghere@UGent.be}
• 关键词：ARS/CEN ; autonomous replicating sequence/centromeric DNA element ; colE1 ori ; colicin E1 origin of replication ; G418 ; geneticin ; GFP ; green fluorescent protein ; OD600 ; optical density measured at 600  ; nm wavelength ; PBS ; phosphate buffered saline ; pHi
• 刊名：International Journal of Food Microbiology
• 出版年：2012
• 期刊代码：63_01681605
• 类别：abs
• 出版时间：1 June, 2012
• 卷：156
• 期：3
• 页码：290-295
• 文件大小：495 K

摘要

It is generally known that intracellular pH (pH_i) plays a vital role in cell physiology and that pH_i homeostasis is essential for normal cellular functions. Therefore, it is desirable to know the pH_i during cell life cycle or under various growth conditions. Different methods to measure pH_i have been developed and among these methods, the use of pH-sensitive green fluorescent protein (GFP) as a pH_i indicator is a promising technique. By using this approach, not only can more accurate pH_i results be obtained but also long-term experiments on pH_i can be performed. In this study, the wild type Zygosaccharomyces bailii, a notorious food spoilage yeast, was transformed with a plasmid encoding a pH-sensitive GFP (i.e. pHluorin), enabling the pH_i of the yeast to be determined based on cellular fluorescent signals. After the transformation, growth and pH_i of the yeast were investigated in four different acidic conditions at 22 掳C during 26 days. From the experimental results, the transformation effectiveness was verified and a good correlation between yeast growth and pH_i was noticed. Particularly, it was observed that the yeast has an ability to tolerate a significant pH_i drop during exponential phase and a subsequent pH_i recovery in stationary phase, which may underlie the exceptional acid resistance of the yeast. This was the first time that a GFP-based approach for pH_i measurement was applied in Z. bailii and that the pH_i of the yeast was monitored during such a long period (26 days). It can be expected that greater understanding of the physiological properties and mechanisms behind the special acid resistance of the yeast will be obtained from further studies on this new yeast strain.