Date of Award

Spring 5-11-2016

Document Type

Thesis

Degree Name

Bachelor of Arts

Department

Biology

First Advisor

Joan L. Slonczewski

Abstract

Escherichia coli are dynamic bacteria that possess the ability to grow and survive across a wide range of external pH conditions. E. coli can grow reasonably well up to ~pH 9.0 and can survive (but not grow) up to ~pH 9.8. We conducted a high pH laboratory evolution experiment in which we grew E. coli at the upper limits of alkali growth in buffered LB media supplemented with potassium. The pH of the culture media began at pH 9.0, but after 464 generations the pH was increased to pH 9.2, then to pH 9.25 after 1,187 generations, and finally to pH 9.3 after 1,901 generations. After 2,187 generations, the experiment was terminated, and eight strains from four populations were purified and sequenced. All populations demonstrated an increase in the endpoint optical density after 22 hours of growth in comparison to the ancestral strain, yet only four out of the eight evolved strains exhibited an increase in growth rate.

In order to better understand the increase in fitness on a genetic basis, mutations in the eight evolved strains were predicted using the breseq computational pipeline. We hypothesize that mutations to sigma factor rpoS may be in part responsible for adaptation given that similar mutations occurred independently across multiple populations. One of the ways E. coli is able to thrive under stressful conditions is by upregulating RpoS, a stress response regulon responsible for the regulation of approximately 10% of its genome. Although much is known about the signaling induction of RpoS under nutrient starvation, acid stress, and other stressful conditions, little is known about the signaling induction of rpoS under high pH stress. We have found that RpoS is induced during growth in high external pH and is stabilized by a combination of the antiadaptors, IraM, IraP, and IraD.

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