RUI: Benzoate and pH Stress in Experimental Evolution of Escherichia coli

在所有环境中，细菌都会经历pH值的变化。低pH值放大了细菌对食物中有机酸的吸收，如与阿司匹林相关的化合物苯甲酸盐和水杨酸盐。对苯甲酸胁迫和适应的研究有助于深入了解微生物与富含水杨酸盐的食用植物(如水果和浆果)的相互作用。植物水杨酸盐是胁迫植物相关微生物群落中细菌的重要防御分子。在这个项目中，本科生研究人员将调查大肠杆菌K-12对酸增强的苯甲酸盐或水杨酸盐吸收的反应。初步结果显示，经过几代人的苯甲酸盐暴露，细菌经历了令人惊讶的适应，包括丧失抗药性，更持久的细胞形状，以及各种有机化合物的产生改变。苯甲酸适应克隆显示了对突变的选择，这些突变增强了对有机酸的耐受性，但代价是对氯霉素等抗菌剂的敏感性。本科生研究人员将进行遗传分析，以测试适应性的增加和对氯霉素的敏感性是否与有机酸诱导的基因网络的丧失有关，或者适应性的变化是否源于以前未知的机制。提高适合度的一个可能机制是失去耗能基因的表达，以克服苯甲酸耗尽质子动能所造成的低能量压力。低能量压力的作用将通过使用耗尽质子动力的解偶联分子(如CCCP)处理，以及通过测试细菌对高pH的反应来测试，高pH是一种导致低能量压力的不同条件。苯甲酸适应克隆的另一种机制可能涉及改善pH动态平衡。本科生将使用比率荧光显微镜测量细菌细胞质的pH值，方法是PI实验室使用的方法，并应要求提供给其他实验室。苯甲酸适应的克隆也表现出细胞形态的变化。本科生研究人员将测试突变等位基因在酸性或碱性条件下维持细胞形状的作用。不寻常的细胞形状表型可以提供关于细胞分裂机制的线索，这是一个对人类控制微生物群落很重要的过程。

In all environments, bacteria experience change in pH. Low pH amplifies the bacterial uptake of organic acids found in food, such as the aspirin-related compounds benzoate and salicylate. Study of benzoate stress and adaptation yields insights about microbial interactions with salicylate-rich food plants such as fruits and berries. Plant salicylates are important defense molecules which stress the bacteria of plant-associated microbial communities. For this project, undergraduate researchers will investigate how the bacterium Escherichia coli K-12 responds to acid-enhanced uptake of benzoate or salicylate. Preliminary results show that over generations of benzoate exposure, bacteria undergo surprising adaptations including the loss of drug resistance, more durable cell shape, and altered production of a variety of organic compounds. Benzoate-adapted clones show selection for mutations that enhance tolerance of organic acids at the cost of sensitivity to antimicrobials such as chloramphenicol. Undergraduate researchers will perform genetic analysis to test whether the fitness gains and chloramphenicol sensitivity are associated with loss of organic acid-induced gene networks, or whether the fitness changes derive from previously unknown mechanisms. A possible mechanism of fitness increase is the loss of energy-expensive gene expression, to overcome the low-energy stress caused by the benzoate depletion of protonmotive force. The role of low-energy stress will be tested by treatment with uncoupler molecules such as CCCP that deplete proton motive force; and by testing bacterial responses to high pH, a different condition that incurs low-energy stress. Another mechanism of benzoate-adapted clones may involve improvement of pH homeostasis. Undergraduates will measure bacterial cytoplasmic pH using ratiometric fluorescence microscopy, by a method used in the PI's lab and provided on request to other labs. Benzoate-adapted clones also show changes in cell morphology. Undergraduate researchers will test the role of mutant alleles in maintaining cell shape in acid or in base. Unusual cell shape phenotypes can yield clues as to mechanisms of cell division, a process important for human control of microbial communities.