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...