Pandey Rachna, Vischer Norbert O E, Smelt Jan P P M, van Beilen Johan W A, Ter Beek Alexander, De Vos Winnok H, Brul Stanley, Manders Erik M M
Molecular Biology and Microbial Food Safety, SILS, University of Amsterdam, Amsterdam, The Netherlands.
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.
Appl Environ Microbiol. 2016 Oct 14;82(21):6463-6471. doi: 10.1128/AEM.02063-16. Print 2016 Nov 1.
Intracellular pH (pH) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pH homeostasis. Unfortunately, accurate pH 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 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 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 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 regulation 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.
细胞内pH值（pH）对细菌细胞生理功能有至关重要的影响。因此，多种食品保鲜策略旨在扰乱pH值稳态。遗憾的是，使用现有方法进行精确的pH值定量并不理想，因为测量的是细胞群体的平均值，没有考虑个体间的异质性。然而，众所周知，同基因群体中的生理异质性是导致细胞在应对外部应激源时生长和分裂动力学差异的原因。为了在这种情况下评估细胞内酸度的行为，我们开发了一种可靠的方法来定量枯草芽孢杆菌单细胞水平的pH值。芽孢杆菌会使食物变质、引发疾病，并且以其形成高度抗逆性孢子的能力而闻名。我们使用了一种改进版的基因编码比率型pH荧光蛋白（IpHluorin），在外部pH值为6.4的条件下，对单独培养的枯草芽孢杆菌细胞在有无弱酸胁迫的情况下进行了pH值定量。在存在3 mM山梨酸钾的情况下，观察到生长细胞的pH值下降以及世代时间增加。当细胞受到25 mM醋酸钾胁迫时也观察到了类似的效果。对生长菌落中的单个细菌进行时间分辨分析表明，在pH值短暂下降后，长期的pH值变化高度依赖于细胞。单细胞水平的异质性表明存在可能更具抗性并有助于群体存活的亚群。我们的方法有助于理解单个细菌中的pH值调节，并可能有助于审查现有和新型食品保鲜策略的效果。
本研究展示了如何在单细胞水平上测量对常用的弱有机酸食品防腐剂（如山梨酸和乙酸）的生理反应。这些数据是将添加弱有机酸食品防腐剂后经常观察到的单细胞异质生长行为联系起来的关键。一般来说，这些数据是以与酸一起孵育的样品平板计数的形式收集的。在这里，我们可视化了细胞pH值稳态中潜在的异质性，为弱酸应激反应异质性的机制分析开辟了道路。因此，微生物风险评估可以变得更加可靠，扩大这些知名弱有机酸食品防腐剂的使用范围。
