We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species Escherichia coli and three gram-positive species, Listeria monocytogenes, Listeria innocua, and Bacillus subtilis. All cells exhibited some degree of physiological damage, but optically trapped E. coli and L. innocua cells and a subpopulation of L. monocytogenes cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of Listeria monocytogenes cells exhibited signs of physiological damage even while trapped at 6 mW, as did B. subtilis cells. Increasing the laser power to 18 mW caused the pH gradient of both Listeria and E. coli cells to decrease within minutes. Moreover, both species of Listeria exhibited morepronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.

We describe the dynamics of changes in the intracellular pH (pH i ) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH ex ). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH i within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH ex from 7.0 to 5.0. Under these conditions, the pH i of L. innocua remained neutral (between 7 and 8). In contrast, the pH i values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH ex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH i at pH ex 7.0, while different kinetics of pH i regulation were observed in different species and also in different strains of S. thermophilus.Bacteria have developed different ways to withstand stressful situations, such as a decrease in the pH ex . Neutrophilic bacteria like Escherichia coli maintain a pH i that is close to neutral when the pH ex is decreased and therefore generate large proton gradients (28). Among the gram-positive bacteria, strains of Enterococcus hirae which were originally identified as Streptococcus faecalis (12) have been studied extensively in order to examine pH homeostasis (14-16). These bacteria also grow at alkaline pH values, and they are considered neutrophiles (31), although they are phylogenetically related to streptococci and lactococci.Many acid-tolerant fermentative bacteria have developed another strategy; in these organisms the pH i decreases as the pH ex decreases during growth (4, 23) in order to maintain a constant pH gradient rather than a constant pH i . Generating a large proton gradient is disadvantageous for fermentative lactic acid bacteria, because proton translocation consumes energy (16), and anaerobic organisms gain significantly less energy from sugar metabolism than aerobes gain. Furthermore, a large proton gradient results in accumulation of organic acid anions in the cytosol (33).Food fermentations are often carried out by sequential microbial populations; this occurs in dairy fermentations, such as yogurt fermentation (32), as well as in indigenous spontaneous fermentations of cereals and vegetables (7,10,20). Lactic acid bacteria, particularly lactobacilli, which are considered the most acid-tolerant bacteria, are often dominant at the end of these fermentations (13, 34). The acid tolerance of these organisms is advantageous, as they have a competitive advantage over known pathogens and other undesirable bacteria when the concent...

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The development of a rapid method for measuring intracellular pH (pH,) in single bacterial cells is described. Lactobacillus delbrueckii subsp. bulgaricus and Listeria innocua were used as test organisms. The method is based upon fluorescence microscopy and ratio imaging of cells stained with carboxyfluorescein succinimidyl ester. After staining, the bacteria were immobilized on a membrane filter and transferred to a closed perfusion chamber, allowing control of the cell environment during analysis. The set-up was optimized with regard to the use of neutral-density filters and background subtraction, for determining the excitation ratio 490 nm/435 nm (R49,,,435) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures.To study the pH, of Lb. delbrueckii subsp. bulgaricus and L. innocua in response to different extracellular pH (pH,,) values, an in vivo calibration curve was constructed in the pH, range 5-0-8-5. Distinct differences between the two cultures were observed. L. innocua maintained a near-neutral pH, almost independently of pH, (5-&8-0), whereas the pH, of Lb. delbmeckii subsp.bulgaricus decreased with decreasing pH, ,. In pure and mixed cultures a t pH, 5-0, the pH, values of Lb. delbmeckii subsp. bulgaricus and L. innocua were 6120.2 and 7.5202, respectively. This difference in pH, may explain how Lb. delbrueckii subsp. bulgaricus obtains a competitive advantage over L. innocua at low pH, , .Keywords : intracellular pH, ratio imaging, single cells, Lactobacillus delbrueckii subsp. bulgaricus, Listeria innocua INTRODUCTIONMost bacteria maintain an intracellular pH (pH,) close to neutral within fairly narrow limits (Padan et al., 19Sl), because this enables metabolic reactions to occur even under unfavoura ble extracellular pH (pH,,) conditions. Bacteria can be divided into three groups with regard to pH homeostasis : neutrophiles, acidophiles and alkalophiles. These groups differ in requirement for pH,,, but all groups maintain a pH, between 6.5 and 9-5 (White, 1995). Acidophiles maintain a large gradient between pH, and pH,, (ApH), but are restricted to growth in very acidic environments due to an inverted membrane potential (White, 1995 group of acid-tolerant fermentative bacteria grows at pH values ranging from neutral to pH 3-5 (Kashket, 1987;McDonald et al., 1990; Russell, 1991a). This group consists of certain ruminal bacteria (Russell, 1991b) and various species of lactic acid bacteria (Kashket, 1987). A common feature is the ability to decrease their pH, with pH,, during growth (Russell & Hino, 1985;Nannen & Hutkins, 1991a;Cook & Russell, 1994), and therefore this group of bacteria does not comply with the conventional classification of pH homeostasis. The pronounced organic acid production of these bacteria creates an environment unfavourable for most other organisms (Russell, 1992), which is the basis of many methods of food preservation by fermentation.Food fermentations are often carried out by a concerte...

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H ؉ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H ؉ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs

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...

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Applying a newly developed user-interactive optical trapping system, we controllably surrounded individual cells of one yeast species, Hanseniaspora uvarum, with viable cells of another yeast species, Saccharomyces cerevisiae, thus creating a confinement of the former. Growth of surrounded and non-surrounded H. uvarum cells was followed under a microscope by determining their generation time. The average generation time of surrounded H. uvarum cells was 15% higher than that of non-surrounded cells, thereby showing that the confinement imposed by viable S. cerevisiae cells on H. uvarum inhibits growth of the latter. This study is the first to demonstrate that confinement is a determinant of growth in a microbial ecosystem.