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值会增加细菌对食物中有机酸的吸收，例如与阿司匹林相关的化合物苯甲酸酯和水杨酸酯。对苯甲酸盐胁迫和适应的研究使人们对富含水杨酸的食物植物（如水果和浆果）的微生物相互作用有了新的认识。植物水杨酸盐是植物相关微生物群落中重要的胁迫细菌的防御分子。在这个项目中，本科生研究人员将研究大肠杆菌K-12如何对酸增强的苯甲酸盐或水杨酸盐摄取作出反应。初步结果表明，经过几代苯甲酸酯的暴露，细菌经历了令人惊讶的适应，包括失去耐药性，更持久的细胞形状，以及改变了各种有机化合物的生产。适应苯甲酸盐的克隆显示出对增强有机酸耐受性的突变的选择，其代价是对氯霉素等抗菌剂的敏感性。本科研究人员将进行遗传分析，以测试适应度的增加和氯霉素敏感性是否与有机酸诱导的基因网络的丧失有关，或者适应度的变化是否源于以前未知的机制。适应度增加的可能机制是能量昂贵基因表达的丧失，以克服苯甲酸酯耗尽原动力引起的低能量应激。低能应力的作用将通过解偶联分子（如消耗质子动力的CCCP）进行处理来测试；并通过测试细菌对高pH值的反应，这是一种不同的条件，会产生低能量压力。适应苯甲酸盐的克隆的另一个机制可能与pH稳态的改善有关。本科生将使用比例荧光显微镜测量细菌细胞质pH值，该方法在PI实验室中使用，并根据要求提供给其他实验室。适应苯甲酸盐的克隆也显示出细胞形态的变化。本科研究人员将测试突变等位基因在酸或碱中维持细胞形状的作用。不寻常的细胞形状表型可以为细胞分裂机制提供线索，这是人类控制微生物群落的重要过程。

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.