Dissertation
DEVELOPMENT AND APPLICATION OF FUNCTION-DRIVEN CHEMICAL BIOLOGY APPROACHES TO CHARACTERIZE CHITINOLYSIS IN SOIL MICROBIOMES
Washington State University
Doctor of Philosophy (PhD), Washington State University
01/2022
DOI:
https://doi.org/10.7273/000004611
Handle:
https://hdl.handle.net/2376/122795
Abstract
Microbial catabolism of chitin plays fundamental roles in ecosystem functioning, yet a full, mechanistic understanding remains elusive. To gain a function-dependent understanding we integrated omics-based techniques with novel chemical biology approaches to directly characterize chitinase activity in a soil biosystem. Herein, the strategies of chemical biology and chemoproteomics, synthesis of activity-based probes, and techniques to measure enzymatic activities in a soil rhizosphere is introduced in Chapter One. A connection is made to show how these function-forward approaches can be applied in reduced complexity soil microbiomes. Chapter Two presents the synthesis of activity-based probes for chitinases and related glycoside hydrolase enzymes by modifying N-acetyl glucosamine and chitotriose. These probes were used to determine the active complement of chitinolytic enzymes produced over time by the soil bacterium Cellvibrio japonicus. Chapter Three discuss the development of stable and simple chitinolytic consortia from native soil. We investigated the successional dynamics of a native soil microbiome during long-term enrichment on chitin and N-acetylglucosamine. We examined succession of the community in a structured soil and unstructured liquid environment at various initial species richness. We found that lower initial richness results in a more rapid convergence to community stability. The structured soil environment, rather than liquid, formed minimized chitinolytic communities best representative of a complex natural soil community. The results provided enhanced understanding about the succession, selection, and stabilization of a chitinolytic microbiome. Chapter Four discusses the application of chemical biology in a soil rhizosphere. We synthesized a fluorescence substrate, chitotriose-TokyoGreen, which is selectively triggered by active chitinase enzymes producing a fluorescence signal to spatially resolve chitinases. When proteins were extracted from a switchgrass rhizosphere using a nitrocellulose membrane and stained with chitotriose-TokyoGreen, chitinases were found to be primarily distributed along an area with supplemented chitin. Finally, Chapter Five provides a summary of the research, describes key scientific contributions, and describes future directions. Overall, our chemical biology approach provides a function-resolved molecular level insight to the roles chitinase and chitinolytic microbes play in soil nutrient cycling in the face of environmental changes.
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Details
- Title
- DEVELOPMENT AND APPLICATION OF FUNCTION-DRIVEN CHEMICAL BIOLOGY APPROACHES TO CHARACTERIZE CHITINOLYSIS IN SOIL MICROBIOMES
- Creators
- Elias Zegeye
- Contributors
- Aaron Wright (Advisor)Xiao Zhang (Committee Member)Alla Kostyukova (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 171
- Identifiers
- OCLC#: 1365399169; 99900901336601842
- Language
- English
- Resource Type
- Dissertation