Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

Abstract

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pH i ) 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 pH i , allowing fast and noninvasive determination of pH i that is ideally suited for direct analysis of individual bacterial cells present in complex environments.Bacteria are often subjected to various forms of environmental stress, and in recent years, there has been increasing focus on the different mechanisms they employ to protect themselves against these environmental changes. One of the most extensively studied stress responses is the ability of bacteria to survive low pH by adjusting their intracellular pH (pH i ) in response to changes in extracellular pH (pH ex ) (1, 4). Several methods have been developed for measuring bacterial pH i (2,14,17,19). However, as these techniques require radioactive labeling and time-consuming staining procedures, noninvasive methods for continuous measurement of pH i in bacteria are in demand. Since the discovery of green fluorescent protein (GFP), a wide range of mutant variants has been created. In eukaryotic cells, several GFP variants with altered excitation and emission spectra have been examined for their use as pH i probes (8,11,12,13,16). One of these GFP variants, ratiometric GFP, was obtained by introducing specific amino acid substitutions to the chromophore, causing the resulting protein to alter its excitation spectrum according to the pH of the surrounding environment (13).In order to express the ratiometric GFP protein in both gram-positive and gram-negative bacterial cells, we inserted the corresponding gene downstream of the P32 promoter in the chloramphenicol-resistant expression vector pMG36c, which replicates in both cell types (20). The ratiometric gfp gene (GenBank accession no. AF058694) was amplified by PCR by using the oligonucleotides C-GFP (5Ј-TAT CCC AAG CTT TTA TTT GTA TAG TTC ATC CAT GCC ATG TG-3Ј) and N-GFP (5Ј-TGC TCT AGA GTA ATA AGG AGG AAA AAA TAT GAG TAA AGG AGA AGA ACT TTT CAC TGG AGT TGT CCC-3Ј) (DNA Technology, Å rhus, Denmark) that additionally introduced an initiating ATG codon as well as a ribosomal binding site (5). The resulting 750-bp DNA fragment was inserted into the XbaI-and EcoRI-digested pMG36c, and the correct DNA sequence of the ratiometric gfp gene present in the resulting plasmid (pGFPratiometric) was confirmed by DNA sequence analysis (data not shown).When we introduced pGFPratiometric in the gram-positive bacterium Lactococcus lactis subsp. lactis CNRZ 157 as previously described (9) and grew cells at 30°C in M17 broth containing 0.5% (wt/vol) glucose and 5 g of chloramphenicol per ml, we found that excitation at 410 nm gave a strong pHdependent fluorescent signal. Furthermore, a pH-independent isosbestic point was seen at 430 nm, which is in accordance with previous results obtained in mammali...

pH of the Cytoplasm and Periplasm of Escherichia coli : Rapid Measurement by Green Fluorescent Protein Fluorimetry

“…1A and B). Previously, GFP has been used as a probe for determination of bacterial pH by fluorescence microscopy but not by fluorimetry of cell suspensions (22). Cells of strains MC4100AR ⌬tatABCDE TorA-GFPmut3* and JLS0617 (W3110/pSL38_YFP) were suspended in buffered M63 medium with supplements, adjusted to pH values in the range from pH 5.5 to 8.0.…”

“…In previous reports, the pH-dependent changes in fluorescence have been observed using fluorescence microscopy (22). Microscopy, however, necessitates cumbersome quantitation procedures that introduce error and limit the time scale of the observable signal.…”

“…Fluorescent proteins provide highly sensitive detection, do not require indicator loading, and lack phototoxicity (5,11,20,23). Derivatives of the green fluorescent protein (GFP) and yellow fluorescent protein (YFP) are pH indicators ideally suited for kinetic studies (11,13,15,22,24). After acidification of the protein's environment, the fluorescence signals of both GFP and YFP decrease in less than 1 ms, allowing rapid detection of pH change (11,15).…”

“…The advent of highly pH-sensitive fluorescent proteins offers new possibilities for measuring cellular pH (11,13,16,22,24). Fluorescent proteins provide highly sensitive detection, do not require indicator loading, and lack phototoxicity (5,11,20,23).…”

Single-Cell Microbiology: Tools, Technologies, and Applications

“…These authors found that small changes in the concentration of CheY-P led to large changes in the rotational bias of the flagellar motor, suggesting that the motor itself acts as a signal amplifier and that additional cellular mechanisms exist for maintaining CheY-P concentrations within the operational range of the motor (58). Other applications of GFP include the construction of whole-cell sensors for in situ monitoring of iron availability on leaf surfaces (120); measurement of cytoplasmic viscosity and protein diffusion rates in living cells (74,182); investigation of quorum-based interspecies communication or coordinated, multicellular behaviors (11,116,126,248); measurement of the internal pH of bacterial cells (172); and real-time reporting of fungal susceptibility to antimicrobial compounds (247).…”