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. 1996 Jan;62(1):178–183. doi: 10.1128/aem.62.1.178-183.1996

A Novel Method for Continuous Determination of the Intracellular pH in Bacteria with the Internally Conjugated Fluorescent Probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester

P Breeuwer, J Drocourt, F M Rombouts, T Abee
PMCID: PMC1388751  PMID: 16535209

Abstract

A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.

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Selected References

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  1. Allen C. N., Harpur E. S., Gray T. J., Simmons N. L., Hirst B. H. Efflux of bis-carboxyethyl-carboxyfluorescein (BCECF) by a novel ATP-dependent transport mechanism in epithelial cells. Biochem Biophys Res Commun. 1990 Oct 15;172(1):262–267. doi: 10.1016/s0006-291x(05)80203-7. [DOI] [PubMed] [Google Scholar]
  2. Booth I. R. Regulation of cytoplasmic pH in bacteria. Microbiol Rev. 1985 Dec;49(4):359–378. doi: 10.1128/mr.49.4.359-378.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Breeuwer P., Drocourt J. L., Rombouts F. M., Abee T. Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry. Appl Environ Microbiol. 1994 May;60(5):1467–1472. doi: 10.1128/aem.60.5.1467-1472.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruno M. E., Kaiser A., Montville T. J. Depletion of proton motive force by nisin in Listeria monocytogenes cells. Appl Environ Microbiol. 1992 Jul;58(7):2255–2259. doi: 10.1128/aem.58.7.2255-2259.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chaillet J. R., Boron W. F. Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. J Gen Physiol. 1985 Dec;86(6):765–794. doi: 10.1085/jgp.86.6.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Graber M. L., DiLillo D. C., Friedman B. L., Pastoriza-Munoz E. Characteristics of fluoroprobes for measuring intracellular pH. Anal Biochem. 1986 Jul;156(1):202–212. doi: 10.1016/0003-2697(86)90174-0. [DOI] [PubMed] [Google Scholar]
  7. James-Kracke M. R. Quick and accurate method to convert BCECF fluorescence to pHi: calibration in three different types of cell preparations. J Cell Physiol. 1992 Jun;151(3):596–603. doi: 10.1002/jcp.1041510320. [DOI] [PubMed] [Google Scholar]
  8. Kashket E. R. The proton motive force in bacteria: a critical assessment of methods. Annu Rev Microbiol. 1985;39:219–242. doi: 10.1146/annurev.mi.39.100185.001251. [DOI] [PubMed] [Google Scholar]
  9. Kondo M., Araie M. Movement of carboxyfluorescein across the isolated rabbit iris-ciliary body. Curr Eye Res. 1994 Apr;13(4):251–255. doi: 10.3109/02713689408995785. [DOI] [PubMed] [Google Scholar]
  10. Lyons A. B., Parish C. R. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131–137. doi: 10.1016/0022-1759(94)90236-4. [DOI] [PubMed] [Google Scholar]
  11. Molenaar D., Abee T., Konings W. N. Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator. Biochim Biophys Acta. 1991 Nov 14;1115(1):75–83. doi: 10.1016/0304-4165(91)90014-8. [DOI] [PubMed] [Google Scholar]
  12. Molenaar D., Bolhuis H., Abee T., Poolman B., Konings W. N. The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis. J Bacteriol. 1992 May;174(10):3118–3124. doi: 10.1128/jb.174.10.3118-3124.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nedergaard M., Desai S., Pulsinelli W. Dicarboxy-dichlorofluorescein: a new fluorescent probe for measuring acidic intracellular pH. Anal Biochem. 1990 May 15;187(1):109–114. doi: 10.1016/0003-2697(90)90425-9. [DOI] [PubMed] [Google Scholar]
  14. Noël J., Tejedor A., Vinay P., Laprade R. Fluorescence measurement of intracellular pH on proximal tubule suspensions. The need for a BCECF sink. Ren Physiol Biochem. 1989 Sep-Dec;12(5-6):371–387. doi: 10.1159/000173215. [DOI] [PubMed] [Google Scholar]
  15. Opitz N., Merten E., Acker H. Evidence for redistribution-associated intracellular pK shifts of the pH-sensitive fluoroprobe carboxy-SNARF-1. Pflugers Arch. 1994 Jun;427(3-4):332–342. doi: 10.1007/BF00374542. [DOI] [PubMed] [Google Scholar]
  16. Peña A., Ramírez J., Rosas G., Calahorra M. Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation. J Bacteriol. 1995 Feb;177(4):1017–1022. doi: 10.1128/jb.177.4.1017-1022.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Poolman B., Driessen A. J., Konings W. N. Regulation of solute transport in streptococci by external and internal pH values. Microbiol Rev. 1987 Dec;51(4):498–508. doi: 10.1128/mr.51.4.498-508.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Salhany J. M., Yamane T., Shulman R. G., Ogawa S. High resolution 31P nuclear magnetic resonance studies of intact yeast cells. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4966–4970. doi: 10.1073/pnas.72.12.4966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  21. Weston S. A., Parish C. R. New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J Immunol Methods. 1990 Oct 4;133(1):87–97. doi: 10.1016/0022-1759(90)90322-m. [DOI] [PubMed] [Google Scholar]
  22. ten Brink B., Konings W. N. Electrochemical proton gradient and lactate concentration gradient in Streptococcus cremoris cells grown in batch culture. J Bacteriol. 1982 Nov;152(2):682–686. doi: 10.1128/jb.152.2.682-686.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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