ORCID

http://orcid.org/0000-0001-7087-6129

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Siderophores are small molecules created by fungi and bacterial cells to help obtain iron. This may extend to cells in iron-limiting environments within host organisms. Certain siderophores are associated with increased virulence in urinary tract infections (UTIs). Siderophore system activity is typically regulated by feedback inhibition via ferric uptake regulator (Fur) which, in the presence of cytosolic iron, binds to a ~19 base pair inverted DNA repeat (9-1-9) called a Fur box. Once bound to this motif, the iron-Fur complex suppresses siderophore expression when adequate bacterial iron status is achieved. Many organisms produce more than one siderophore, and in the case of certain E. coli strains, up to four. This has been frequently interpreted as an example of biological redundancy, where multiple biological systems are deployed to fulfill a common function, in this case iron acquisition. While this may provide a backup option in the event that one system fails, it would also introduce new metabolic costs to bacteria. In this thesis we have tested the redundancy hypothesis for siderophore systems commonly possessed by Escherichia coli isolates recovered from patients with urinary tract infections. The virulence-associated siderophore yersiniabactin (Ybt) is commonly secreted by infection-associated E. coli isolates and has also been shown to bind a broad range of metal ions in addition to iron (III). Nevertheless, Ybt has been understood to function in the manner of a canonical siderophore. E. coli that secrete Ybt are also capable of secreting the prototypical siderophore enterobactin (Ent). To test the hypothesis that Ent and Ybt are redundant for iron acquisition, we developed an assay that measures siderophore-dependent growth in iron-limited cultures. Contrary to this hypothesis, we found that the ability of E.coli to secrete Ybt does not compensate for the loss of Ent in the test system, and that Ent is uniquely necessary for siderophore-dependent growth. Growth of an Ent-deficient strain was rescued by addition of purified Ybt to the medium, leading us to determine whether Ybt secretion was inadequate to compensate for the absence of Ent. This led to the discovery that, relative to Ent, peak Ybt production is achieved late in the bacterial growth cycle at higher cell density. This is not attributable to Fur regulation alone. We considered that Ybt production is attributable to the density-dependent regulation characteristic of quorum sensing (QS) mechanisms. Consistent with this, we found that Ybt acts as the autoinducer in a QS autoregulatory circuit. In the initial low cell density conditions of our siderophore-dependent growth assay when extracellular Ybt concentrations are low, the Ybt system is less activated than the Ent system and unable to compensate for the absence of Ent. These data are consistent with a regulatory model in which the Ybt system is an autoinducer and YbtA, an AraC-like transcription factor, is the cellular Ybt sensor that activates further Ybt biosynthesis. To our knowledge, this is the first QS mechanism found to control siderophore production in E. coli and the first in which the siderophore plays a dual role as autoinducer. The results suggest a virulence-associated role for Ybt production at high bacterial population densities. We compared another virulent-associated siderophore called aerobactin (Aer) to Ent. When looking at production of siderophores over a bacterial growth curve we observed a delay in Aer production, similar to what is found in Ybt. This potentially suggest that a similar alternative regulation system could apply to Aer. We also tested possible theories about what a potential non redundant function of Aer. We were unable to prove any one function but have found a possible connection between Aer and antibiotic resistance.

Language

English (en)

Chair and Committee

Jeffrey Henderson

Committee Members

Daniel Goldberg, Michael Gross, Christina Stallings, Joseph Vogel,

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