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Published on: January 17, 2014
Intracellular pH determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis
P F X Corvini1, H Gautier1, E Rondags1
1Laboratoire de Fermentations et de Bioconversions Industrielles1, and Laboratoire des Sciences du Génie Chimique - CNRS2, Institut National Polytechnique de Lorraine, 2 Avenue de la Forêt de Haye, BP 172, F-54505 Vandoeuvre-lès-Nancy, France.
Abstract:
Intracellular pH (pH(i)) is an essential parameter in the regulation of intracellular processes. Thus, its measurement might provide clues regarding the physiological state of cells cultivated in vitro. pH(i) of the filamentous, pristinamycin-producing Streptomyces pristinaespiralis was determined by epifluorescence microscopy and image analysis using the pH-sensitive fluorescent probe BCECF-AM [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD(660)=1) of S. pristinaespiralis with 20 microM BCECF at 28 degrees C for 30 min yielded a green/red fluorescence ratio (R:(527/600)) that correlated with the pH(i) of the cells for values ranging from 6.5 to 8.5. When S. pristinaespiralis was cultivated in pristinamycin-producing conditions (in batch mode, with a constant external pH of 6.8), the measured pH(i) varied between 6.3 and 8.7. In fact, pH(i) correlated with the excretion of pristinamycins and glucose consumption during the production process.
