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Microbiology 146 (2000), 2671-2678
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Microbiology (2000), 146, 2671-2678.
© 2000 Society for General Microbiology

Biotechnology

Intracellular pH determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis

P. F. X. Corvini1, H. Gautier1, E. Rondags1, H. Vivier2, J. L. Goergen2 and P. Germain1

Laboratoire 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

Author for correspondence: J. L. Goergen. Tel: +33 383 59 58 44. Fax: +33 383 59 57 96. e-mail: Jean-Louis.goergen{at}ensaia.inpl-nancy.fr

Intracellular pH (pHi) 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. pHi 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 (OD660=1) of S. pristinaespiralis with 20 µM BCECF at 28 °C for 30 min yielded a green/red fluorescence ratio (R527/600) that correlated with the pHi 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 pHi varied between 6·3 and 8·7. In fact, pHi correlated with the excretion of pristinamycins and glucose consumption during the production process.

Keywords: intracellular pH, Streptomyces pristinaespiralis, epifluorescence microscopy, image analysis

Abbreviations: BCECF-AM, 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester; pHi, intracellular pH; R527/600, green/red fluorescence ratio (527/600 nm)




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