Date of Award

Spring 5-6-2019

Document Type

Thesis

Degree Name

Bachelor of Arts

Department

Biochemistry & Molecular Biology

First Advisor

Dr. Joan Slonczewski

Abstract

PMF is necessary for the production of ATP through oxidative phosphorylation, pH regulation, and export of many types of antibiotics. However, the genetic response to depletion of proton motive force (PMF) over an extended period of time in bacteria is poorly understood. Thus, we conducted an experimental evolution of Escherichia coli K-12 in increasing concentrations of the strong PMF uncoupler carbonyl cyanide m-chlorophenylhydrazone. We serially diluted cultures of E. coli K-12 W3110 1:100 each day at pH 6.5 or 8.0 for 1,000 generations. Concentrations of CCCP increased in a stepwise fashion over the course of the evolution from 20 and 50 µM CCCP for low and high pH conditions respectively to 150 µM CCCP for each pH condition. Whole genome sequence analysis of strains isolated after 1,000 generations revealed loss of function mutations to mprA, the repressor of emrAB (MFS family efflux pump), in nearly all strains sequenced. We also found mutations in cecR, the regulator of cefoperazone and chloramphenicol sensitivity. Artificial deletions of both mprA and cecR, in the ancestral strain results in increased CCCP tolerance, supporting the hypothesis that selection favored increased levels of the EmrAB-TolC multidrug efflux pump which expels CCCP from the cell over other drug pumps, which become downregulated or deleted. These results are consistent with data from a previous experimental evolution project in which increased antibiotic sensitivity arose in benzoate-evolved E. coli. This work may have implications for our understanding of the gut microbiome, which experiences high levels of aromatic membrane-permeant acid stress.

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