Quantification of Escherichia coli alternate electron acceptor gene expression under aerobic conditions using a fluorescent reporter

Date of Award

Spring 2017

Document Type

Restricted Thesis

Terms of Use

© 2017 Luke M. Eppley. All rights reserved.

Degree Name

Bachelor of Arts

Department

Biology Department

First Advisor

Amy Cheng Vollmer

Abstract

Escherichia coli contains membrane bound electron-transport chains, which incorporate various dehydrogenases and terminal reductases used in respiration. While oxygen is the preferred terminal acceptor, as it offers the largest redox potential (and therefore maximizes ATP production), E.coli has been shown to utilize five alternate acceptors when grown anaerobically. Nitrate, nitrite, TMAO, DMSO, and fumarate, listed in descending order of energy conservation, all act as acceptors in order to maintain respiration. Almost all of the reductases associated with each of these molecules are regulated by two-component systems to safeguard against unnecessary energy expenditure when a better electron acceptor is present. Notably, previous research in the Goulian laboratory showed that expression of torC, the gene encoding the TMAO-specific reductase, exhibited remarkable variance in cell-to-cell deactivation in aerobic, TMAO-induced cultures. This resulted in many single cells exhibiting no activation as expected, while others showed markedly high expression. This effect had yet to be studied in the other terminal electron acceptors, Our investigation was undertaken to determine whether this observation was specific to TMAO, or more generalizable to other alternative electron acceptors, such as the nitrate-specific narGHJI and the DMSO-specific dmsABC operons. Using a fluorescent reporter (yfp), attempts were made to introduce promoter fusions (PnarG::yfp, PdmsA::yfp) into the E. coli chromosome. However the method for such a site-specific integration were not successful, Subsequently, PnarG::yfp and PdmsA::yfp constructs were placed on a single-copy plasmid. Cultures containing these plasmids were then grown aerobically and anaerobically to compare mean fluorescence reporting. Unlike the previous torC studies, analysis of fluorescent micrographs showed no significant distribution difference between aerobic and anaerobic activation for both PnarG::yfp and PdmsA::yfp constructs. Moreover, the means between conditions within each construct appeared dissimilar in both instances. This indicates that there may be a difference between the activating mechanisms for these alternative electron acceptors during anaerobic respiration, and that a difference may exist in the oxygen-dependent half of the two-component system compared to the torCAD operon. This discrepancy can be reconciled with an explanation based in the more stringent oxygen- and nitrate- availability sensing mechanisms present within E. coli.

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