Date of Award

1-1-2010

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

P. Lee Ferguson

Abstract

Quorum sensing is a process through which bacterial cells monitor their own population densities by producing, sensing and responding to surrounding chemical signals known as autoinducers. A wide range of Gram-negative Proteobacteria possess LuxR-type transcriptional regulatory proteins which can perceive and bind cognate autoinducer N-acylhomoserine lactones (AHLs), thereby inducing a conformational change and subsequently binding to lux box-like target genes to regulate transcription. The study of the first identified quorum-sensing system, the Vibrio fischeri LuxI/LuxR paradigm, was limited to molecular genetic approaches due to the formation of inclusion bodies when overexpressing LuxR in Escherichia coli. The addition of its native autoinducer, N-(3-oxohexanoyl)-homoserine lactone (3OC6-HSL), during bacterial growth has made it possible to purify soluble, native LuxR from recombinant E. coli. The goal of my research was to investigate the interaction between LuxR and the lux box DNA as modulated by changes in the molecular structure of AHLs in vitro.

Indirect photolysis was performed at acidic and basic pH with N-hexanoyl-homoserine lactone (C6-HSL) to examine how environmental factors (e.g., pH, UV light and oxidants) affect the chemical structures of AHLs. High-performance liquid chromatography coupled to tandem mass spectrometry was employed for structural elucidation with additional diagnostic structural information acquired via in-source collision-induced dissociation. Results indicated that concurrent oxidation and nitration occurred on the acyl side chain of C6-HSL and its open-ring form. The structures of eleven hydroxyl radical oxidation products were postulated. Furthermore, LuxR was copurified with 3OC6-HSL, but extensive dialysis was required to completely remove the ligand from this complex. Dialyzed LuxR was shown to be dimeric and the addition of 3OC6-HSL revealed no change in the oligomeric state. Studies also demonstrated the binding of LuxR to the lux box in a ligand- and concentration-dependent manner. Although the 3-oxo group is not necessary for ligand binding, it affects the oligomeric states of LuxR and the resulting DNA complex. The apparent equilibrium constants of this interaction, obtained through surface plasmon resonance, were used to evaluate the binding affinities of various AHLs for LuxR.

Rights

© 2010, Yiling Cui

Download

Share

COinS