Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells

Abstract

Intracellular pH (pH i ) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pH i homeostasis. Unfortunately, accurate pH i quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pH i at single-cell levels in Bacillus subtilis. Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pH i in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pH i and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pH i regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCEThis study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.

Intracellular pHluorin as Sensor for Easy Assessment of Bacteriocin-Induced Membrane-Damage in Listeria monocytogenes

“…In previous studies, pHluorin was used to investigate membrane homoeostasis of Escherichia coli and Bacillus subtilis following acid stress (Martinez et al, 2012), proton leakage through the MotA/B proton channel of the flagellar apparatus of Salmonella enterica (Morimoto et al, 2010), or the effect of potassium uptake on metabolism of Staphylococcus aureus (Gries et al, 2016). Moreover, pHluorin has been successfully used in B. subtilis both on population and single cell level to study antibacterial effects of weak organic acids to assess their potential as food preservation strategies (Ter Beek et al, 2015; Pandey et al, 2016). In the present study, we investigated the potential of pHluorin to assess membrane-damaging activity of bacteriocins in L. monocytogenes .…”

“…Thus, expression of pHluorin is a convenient approach to identify and test novel antimicrobial compounds active against L. monocytogenes and other (human) pathogens. Additionally, L. monocytogenes EGD-e/pNZ-P help -pHluorin and derivatives in combination with fluorescence ratio imaging microscopy (Martinez et al, 2012; Pandey et al, 2016) can be exploited for infection research to study the mechanisms of pH-homeostasis of L. monocytogenes e.g., in highly acidic phagosomes/phagolysosomes macrophages and other host cells.…”

Adaptation in Bacillus cereus: From Stress to Disease

“…Such flexibility occurs in B. cereus spores ( Van Melis et al, 2014 ). It would be interesting and important to find out whether culture heterogeneity also plays a role in the responses of B. cereus toward acid stress and low oxygen tension ( Pandey et al, 2016 ). Overall, it is expected that investigations of the stress physiology of B. cereus will continue to be central to understanding its behavior in challenging environments.…”

Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis

“…Herein, we found that the residual pyruvate acidified the extracellular pH (pH ex ) and intracellular pH (pH in ), with the lowest pH ex and pH in being 5.7 and 6.0 for BSGN12 during fermentation. The pH in critically affects bacterial cell physiology, such as protein synthesis and enzyme activity [7–10]. The activity of Ce GNA1 is pH-dependent with an optimum pH of 8.2, which is similar to other GNA1 homologues that generally function in alkaline conditions (pH 7.4–9.7), thus it is crucial to maintain intracellular pH homeostasis for the enhanced activity of Ce GNA1 and improved production of GlcNAc [4].…”