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

Kenyon College, Gambier OH

Investigators

Abstract

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.