Date of Award

5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Ocean Engineering and Marine Sciences

First Advisor

Julia E. Grimwade

Second Advisor

Alan C. Leonard

Third Advisor

Andrew G. Palmer

Fourth Advisor

Eric Guisbert

Abstract

The increasing number of drug resistant pathogenic bacteria has created a growing health care crisis. Solving the crisis will require a number of solutions, among them being development of new antibiotics that target novel cell processes. Initiation of chromosomal DNA replication is one such unexploited process. Bacterial cells begin chromosome replication by building a nucleoprotein complex called the orisome which, unwinds replication origin DNA, and loads the replicative helicase. A greater understanding of how orisomes assemble, and how the assembly is regulated, should provide a source of druggable targets to be used in future antibiotic discovery efforts. However, although all bacterial cells use the same initiator protein (DnaA) to assemble the orisome, the replication origins that direct the assembly are more diverse among different bacterial types. Still, because all orisomes must accomplish the same essential tasks, one must speculate that some elements essential for orisome function would be conserved. The goal of this study was to identify some of these potential elements. Using the best studied system for orisome assembly, Escherichia coli, we performed a systematic mutational analysis of the chromosomal E. coli origin of replication, to examine specific elements used in orisome assembly, and to identify features that were essential for triggering the onset of chromosome replication. Three major features were examined: 1) low affinity DnaA-ATP binding sites, 2) the number of low affinity sites, and 3) a region of origin DNA that forms a sharp bend in oriC at the time of initiation. Surprisingly, although ATP-DnaA is considered to be the “active” form or the initiator protein, we found that the DnaA-ADP is capable of performing all of the mechanical functions required for origin activation, if it can bind to the origin of replication. Rather the ATP-bound form of the initiation seems to be required to regulate access of the initiator to the origin, needed to regulate the timing of chromosome replication during the cell cycle. We also found that only a subset of low affinity DnaA recognition sites are required for origin function, although each site appeared to have an incremental role in origin unwinding. Finally, we found that the sharp bend region was essential for E coli origin activity, but this requirement could be suppressed by making the E coli origin resemble the B. subtilis origin, which does not contain the sharp bend region. These studies revealed a surprising plasticity in orisome assembly, and based on our studies, we propose that different bacteria may contain different essential features that direct the mode of origin activation. Features that are shared among bacteria may lead to identification of targets for broad spectrum antibiotics, while type specific essential features could lead to more focused therapies against specific pathogens.

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