Undergraduate Honors Thesis Projects

Date of Award

2023

Document Type

Honors Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biology & Earth Science

Advisor

Dr. Jennifer A. Bennett

First Committee Member

Dr. Jennifer Bennett

Second Committee Member

Dr. John Tansey

Third Committee Member

Dr. Halard Lescinsky

Keywords

Protein Interaction, Cyclic di-GMP, Cell Signaling

Subject Categories

Cell Biology

Abstract

The species Streptomyces coelicolor is a member of a genus that is particularly interesting for antibiotic research in the medical and pharmaceutical industry. S. coelicolor, when exposed to stimuli, enacts the use of a second messenger, called cyclic di-GMP, which signals for the natural production of antibiotics, biofilm formation, life cycle progression, and several other major functions within the cell. It is believed that the signaling within bacteria is largely dependent on the local concentration of cyclic di-GMP. Effective elimination of individual phosphodiesterases and diguanylate cyclases has been observed to greatly affect the cells of E. coli without changes in the overall level of cyclic di-GMP present, suggesting that each enzyme can interact with the signaling molecule to result in a response.

The major purpose of this research was to isolate and test for interactions between the two major cyclic di-GMP phosphodiesterases (RmdA and RmdB) of S. coelicolor. There were two main approaches to testing these relationships. Addressing the biochemical focus of this project, these proteins have been overexpressed within E. coli. From there, purification of individual proteins allowed for the testing of interactions among RmdA with itself, RmdB with itself, and between the two, using Surface Plasmon Resonance (SPR). The other avenue of this investigation involves a Bacterial Two-Hybrid (BACTH) system, to further confirm these findings and serve as a more visual representation of protein interaction. Previous SPR experiments show an interaction between RmdB and itself, suggesting homodimer formation is important for protein function. Additionally, preliminary BACTH experiments suggest RmdA may also form a homodimer for interaction. The next steps of experimentation will aim to determine whether the formation of a heterodimer occurs as part of this second messenger pathway.

There is still much to understand about the functioning of the cyclic di-GMP pathway in Streptomyces. This project investigates the relationships between cyclic di-GMP metabolizing proteins to further build understanding of the S. coelicolor cyclic di-GMP interactome.

Licensing Permission

Copyright, all rights reserved. Fair Use

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Acknowledgement 2

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