Date of Award

Spring 5-6-2019

Document Type

Thesis

Degree Name

Bachelor of Arts

Department

Biology

First Advisor

Joan Slonczewski

Abstract

A previous experimental evolution of Escherichia coli K-12 W3110 in the presence of the membrane-permeant organic acid, benzoate, produced strains that tolerate 20 mM benzoate but are more sensitive to chloramphenicol than the ancestor. We studied the genetic and transcriptomic changes underlying this phenotype. Whole-genome sequences from early generations of the evolution experiment suggest that downregulating/deleting genes of the acid fitness island is an early adaptation in the presence of benzoate. Transcriptomes of four benzoate-evolved strains revealed that after 2,000 generations, these strains had reversed many of the effects benzoate has on gene expression in the ancestor. This involved downregulating acid tolerance genes, drug efflux pumps, and hydrogenases, while upregulating outer membrane porins. We demonstrated that deleting genes of the acid fitness island or proton-motive-force-consuming drug efflux pumps increases the benzoate-tolerance of the ancestor. Additionally, deleting hydrogenase-3 is advantageous in stationary phase. We then investigated several mutations that appear in only one benzoate-evolved strain, including a missense mutation in RNA-polymerase subunit alpha that confers chloramphenicol sensitivity, as well as the deletion of a stress-response regulator (cspC) which increased benzoate tolerance. In general, the benzoate-evolved strains have inverted the effects of benzoate on the transcriptome, decreasing the expression of acid-stress genes, and drug efflux pumps that consume proton motive force.

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