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...
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
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