Type
Text
Type
Dissertation
Advisor
Boon, Elizabeth M | Drueckhammer, Dale | Tonge, Peter | El-Maghrabi, M. Raafat.
Date
2015-12-01
Keywords
filter-binding assay, H-NOX, Nitric oxide, NO responsive quorum sensing circuit, Quorum sensing, Two-component signaling | Biochemistry
Department
Department of Chemistry.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/77159
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Bacterial quorum sensing (QS) is an essential cell-to-cell communication system used by bacteria. Bacteria produce and detect small signaling molecules called autoinducers (AIs) to assess their surrounding bacterial population. Once the AI concentration reaches a threshold, the entire bacterial population undertakes a coordinated change in gene expression patterns. Thus, QS enables bacteria to orchestrate population-wide changes in behavior appropriate for different growth phases or environmental conditions. QS is widely spread among bacteria and regulates a wide array of biological functions that are critical for their survival. Controlled manipulation of QS may provide the means for the design of more selective antibiotics and improved bioproduction efficiency with engineered bacteria. Thus, understanding mechanisms of quorum sensing is of significant academic, medical and industrial interest. In this study, we report the identification and characterization of a nitric oxide (NO) responsive quorum sensing circuit in the marine pathogen Vibrio parahaemolyticus. We found that V. parahaemolyticus QS is regulated not only by AIs, but also by NO, through two-component signaling. V. parahaemolyticus detects NO through a nitric oxide/oxygen binding protein (H-NOX) and H-NOX regulates the activity of an H-NOX-associated histidine kinase (HahK). HahK ultimately modulates the activity of the QS master regulator, OpaR, through phosphoryl group transfer, and thus H-NOX/HahK participates in the V. parahaemolyticus QS circuit. In this thesis, we also present the development of a medium-throughput filter-binding assay for histidine kinase phosphorylation. A major bacterial signaling pathway, two-component signaling, is based upon phosphorylation and phosphotransfer events by histidine kinases and their partner proteins. In spite of its importance, understanding of two-component signaling is hindered by the lack of effective assay methods to quantify histidine kinase activity. To address this issue, we developed a carefully optimized filter-binding assay for kinase phosphorylation detection. We demonstrate the validity of the assay by monitoring phosphorylated histidine degradation under the proposed experimental conditions. Subsequently, we quantify kinase activities with respect to time, enzyme, and substrate concentrations, then determine their kinetic parameters. | 101 pages
Recommended Citation
Ueno, Takahiro B., "Characterization of bacterial multi-component regulatory systems: Nitric oxide, quorum sensing and" (2015). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2995.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2995