Date of Graduation

5-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Poultry Science (PhD)

Degree Level

Graduate

Department

Poultry Science

Advisor/Mentor

Kwon, Young

Committee Member

Graham, Danielle

Second Committee Member

Sun, Xiaolun

Third Committee Member

Huang, Yan

Keywords

chicken gut microbiome; community functional genomics; metagenome-assembled genomes; Tn-seq; transposon delivery

Abstract

The chicken gut microbiome comprises hundreds of bacterial species whose functional roles remain largely unknown, in part because the tools needed to experimentally manipulate these organisms in their native community context have not been systematically developed or validated. This dissertation addresses the gap through three connected research chapters that progressively build a framework for transposon-based genetic delivery in the chicken cecal microbiome, moving from community-wide accessibility mapping to vector optimization to library preparation refinement. In Chapter 2, we established the community-level insertion landscape in chicken cecal metagenome using Himar transposon delivery via RP4 conjugation and aligned Tn-seq reads to a reference database of 12,339 metagenome-assembled genomes (MAGs), for which insertion patterns were characterized across three biological replicates. We found mapping efficiencies were consistent (13.42%-14.54%), but unique insertion sites plateaued despite increasing sequencing depth, indicating library saturation with a reads-per-insertion ratio rising from 6.34 to 21.15. Insertion events were strongly non-uniform, concentrating in a small cohort of dominant MAGs, particularly within Escherichia, Lacticaseibacillus, and Limosilactobacillus. Gram classification had no significant bearing on transposon accessibility (Wilcoxon p = 0.73), while oxygen dependency did, with facultative anaerobes dominating the insertion landscape in a pattern that mirrors the physiology of the anaerobic cecal environment. Reproducibility improved markedly when data were aggregated from MAG-level (ρ = 0.70-0.76) to genus-level (ρ = 0.81-0.84), suggesting that variability in the distribution of transposon insertions at the strain level decreased at higher taxonomic levels, as expected, highlighting the reproducible metagenome-wide insertion profiles. In Chapter 3, we used barcoded pGT plasmids for successful tracking of each transposon insertion, in which a pool of 13 barcoded pGT Himar plasmids was delivered conjugatively into the chicken caecum, with transconjugants confirmed by flow cytometry at 9.2%-13.3% across replicates. Of the 13 constructs, only pGT-Kh2 and pGT-Ah2 consistently dominated across all recovery metrics, read abundance, CPM normalization, insertion efficiency (60%-80%), MAG recovery, and genus-level representation, with the remaining constructs contributing negligibly, indicating that regulatory element-recipient compatibility is the principal determinant of vector success. Similarly, our data show that the flow cytometer cell sorting (FACS) of the transconjugant does not improve the final mapping results. In Chapter 4, we demonstrated using pGT-Kh2 that BamHI restriction digestion, which cuts immediately outside of the inverted repeat on the right end of the plasmid, prior to Tn-seq library preparation, dramatically improved data quality: donor-derived reads were reduced from approximately 25% to under 2%, mapped reads increased 5-6-fold, and reads per insertion improved 4-5-fold, while preserving the distribution patterns of insertion events across metagenomes (Spearman ρ > 0.90 across all replicates). Together, these three studies provide a progressive methodological and biological framework for metagenomeic-level functional genomics in the chicken gut microbiome, identifying the potential community members most tractable for genome editing, the most effective delivery vectors customized to a given taxonomy, and the most reliable library preparation strategy for more comprehensive and reliable metagenome-wide transposon insertion profiling in this ecologically consequential system.

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