Noninvasive Measurement Of Bacterial Intracellular PH On A Single ...

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Abstract

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.

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Figures

FIG. 1.

FIG. 1.

Correlation between pH i and…

FIG. 1.

Correlation between pH i and R 410/430 of ratiometric GFP in pH-equilibrated cells. …

FIG. 1. Correlation between pHi and R410/430 of ratiometric GFP in pH-equilibrated cells. L. lactis and E. coli single cells were equilibrated with 10 kIU of nisin ml−1 (closed circles) and 10 μM CCCP (closed triangles), respectively. Each point represents the mean value for 20 individual cells, with error bars indicating the standard deviations.
FIG. 2.

FIG. 2.

Determination of L. lactis pH …

FIG. 2.

Determination of L. lactis pH i as a function of pH ex by…

FIG. 2. Determination of L. lactis pHi as a function of pHex by use of ratiometric GFP and cFDAse. The pHi of cells transformed with ratiometric GFP (closed circles) was obtained by measuring the R410/430 and converting the obtained value to the correlating pHi. For validation, pHi was also obtained by staining the cells with cFDAse (open circles) and measuring the R490/435. Each point represents the mean value for 20 individual cells, with error bars indicating the standard deviations.
FIG. 3.

FIG. 3.

Determination of E. coli pH …

FIG. 3.

Determination of E. coli pH i as a function of pH ex .…

FIG. 3. Determination of E. coli pHi as a function of pHex. Cells expressing ratiometric GFP were resuspended in phosphate buffers at pHs ranging from 5.5 to 8.5. The R410/430 was determined and was transformed to the corresponding pHi. Each point represents the mean value for 20 individual cells, with error bars indicating the standard deviations.
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References

    1. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49:359-378. - PMC - PubMed
    1. Breeuwer, P., J.-L. Drocourt, F. M. Rombouts, and T. Abee. 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. Appl. Environ. Microbiol. 62:178-183. - PMC - PubMed
    1. Budde, B. B., and M. Jakobsen. 2000. Real-time measurements of the interaction between single cells of Listeria monocytogenes and nisin on a solid surface. Appl. Environ. Microbiol. 66:3586-3591. - PMC - PubMed
    1. Cook, G. M., and J. B. Russell. 1994. The effect of extracellular pH and lactic acid on pH homeostasis in Lactococcus lactis and Streptococcus bovis. Curr. Microbiol. 28:165-168.
    1. Denoya, C. D., D. H. Bechhofer, and D. Dubnau. 1986. Translational autoregulation of ermC 23S rRNA methyltransferase expression in Bacillus subtilis. J. Bacteriol. 168:1133-1141. - PMC - PubMed
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