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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...

Aims:To optimize the practical use of the bacteriocin producing Leuconostoc carnosum 4010 in order to inhibit the growth of Listeria monocytogenes in sliced meat products. Methods and Results: Four different methods for biopreservation using the partially purified bacteriocin or the living culture of Leuc. carnosum 4010 were evaluated. The methods using the living protective culture added to the sliced gas packed meat product were more effective in preventing growth of L. monocytogenes than the use of the partially purified leucocins 4010 or bacteriocin produced during fermentation before heat treatment of the saveloy. The application method giving the highest reduction in L. monocytogenes used nozzles for sprinkling the protective culture on all surfaces of each slice of the meat product. In the control samples without the protective culture, L. monocytogenes grew to ca. 10 7 CFU g )1 , whereas for the application method using nozzles for distributing the protective culture, counts of L. monocytogenes never exceeded 10 CFU g )1 during 4 weeks of storage at 10°C. Conclusions: The live cells of the bacteriocin producing Leuc. carnosum 4010 was the most efficient method as it inhibited the growth of L. monocytogenes in cooked, sliced and gas packed saveloy stored at 5 and 10°C for 4 weeks. Significance and Impact of the Study: The results indicate that biopreservation with lactic acid bacteria is a suitable alternative to chemical preservatives. An even distribution of the protective culture was found to be essential for the efficacy of the protective culture in pilot plant trials.

Aims: To determine inactivation of Listeria monocytogenes by the two lactic acid isomers. Methods and Results: The survival of four strains with varying sensitivity to acid was determined following treatment with L L-or D D-lactic acid at 100 mmol l )1 (pH 3AE7) or HCl at pH 3AE37. There was some, but not complete, similarity in the relative sensitivity of the four strains to the two types of acid. All strains were most sensitive to D D-lactic acid, which gave 0AE6-2AE2 log units greater reduction than L L-lactic acid midway in the inactivation curves. Even very low concentrations of the two isomers had an immediate effect on pH i which was identical for the two isomers. Conclusions:The results show that L. monocytogenes is more sensitive to D D-than to L L-lactic acid; however, this difference is less than the strain variation in L L-lactic acid sensitivity. Significance and Impact of the Study: This work has implications for the application of lactic acid for food preservation as well as for the understanding of the antibacterial mechanisms of weak organic acids.

A mixed culture of single cells of Listeria monocytogenes and the bacteriocin producing Leuconostoc carnosum 4010 showed growth inhibition of L. monocytogenes, although the intracellular pH (pHi) of L. monocytogenes followed by fluorescence ratio imaging microscopy was not affected. Furthermore, L. monocytogenes was exposed to the bacteriocins leucocins 4010 and nisin either in a liquid filled chamber or on the surface of an agar containing bacteriocins. Both bacteriocins caused dissipation of the pH gradient in L. monocytogenes and the effect was clearly dependent on the matrix, as the decrease in pHi occurred much more rapidly in liquid than in agar.