title: Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells.
abstract: Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHihomeostasis. Unfortunately, accurate pHiquantification 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 pHiat 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 pHiin 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 pHiand 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 pHiregulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies.
  
  
  
  This 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.
authors: Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands.
authors: Rachna
authors: R
authors: Pandey
authors: Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands.
authors: Norbert O E
authors: NO
authors: Vischer
authors: Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands.
authors: Jan P P M
authors: JP
authors: Smelt
authors: Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands.
authors: Johan W A
authors: JW
authors: van Beilen
authors: Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands.
authors: Alexander
authors: A
authors: Ter Beek
authors: Department of Veterinary Sciences, Laboratory of Cell Biology and Histology, Antwerp University, Antwerp, Belgium Department Molecular Biotechnology, Cell Systems and Imaging Group, Ghent University, Ghent, Belgium.
authors: Winnok H
authors: WH
authors: De Vos
authors: Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands s.brul@uva.nl.
authors: Stanley
authors: S
authors: Brul
authors: Van Leeuwenhoek Centre for Advance Microscopy, SILS, University of Amsterdam, Amsterdam, The Netherlands.
authors: Erik M M
authors: EM
authors: Manders
mesh_terms: D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017484:Dermatitis, Phototoxic; D005519:Food Preservation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019347:Potassium Acetate / Q000494:pharmacology; D059010:Single-Cell Analysis; D013011:Sorbic Acid / Q000494:pharmacology*; D013312:Stress, Physiological; D059008:Time-Lapse Imaging
chemical_list: C485187:PHluorin; D049452:Green Fluorescent Proteins; D019347:Potassium Acetate; D013011:Sorbic Acid