Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

Abstract

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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