Date of Completion

5-7-2020

Embargo Period

5-7-2020

Keywords

Microbiology, Bioinformatics, Molecular Biology, Oxford Nanopore minION, Illumina, Trachymyrmex septentrionalis, Pseudonocardia, antibiotics, natural products, bioassay

Major Advisor

Dr. Jonathan Klassen

Associate Advisor

Dr. Joerg Graf

Associate Advisor

Dr. Marcy Balunas

Associate Advisor

Dr. Spencer Nyholm

Associate Advisor

Dr. Sarah Hird

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The focus of this thesis is on the genomic diversity of a fungus-growing ant Trachymyrmex septentrionalis symbiont, the actinomycete bacterium Pseudonocardia. Pseudonocardia symbionts engage in host defense via the production of antimicrobial compounds that protect the symbiosis against fungal pathogens that attack the ant’s food source, pathogens that attack the ant hosts, and more generalized pathogens. These compounds are encoded by biosynthetic gene clusters (BGCs) in the genome of these Pseudonocardia and comprise an untapped resource for drug discovery.

Actinomycete genome sequencing is a key component of modern antimicrobial drug discovery efforts. Short-read sequencing leads to genome fragmentation as a consequence of short read lengths that are unable to resolve repetitive BGCs. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate read lengths >2,000,000 base pairs that significantly improve assembly contiguity. However, MinION sequencing is prone to indel and homopolymer errors, necessitating error correction after genome assembly. This work validates a hybrid sequencing protocol that combines long-read ONT MinION data polished by high-accuracy Illumina reads to provide reliable and contiguous assemblies that can be applied broadly for sequencing and assembly of actinobacterial genomes for antimicrobial discovery.

To leverage Pseudonocardia symbionts for drug discovery, a better understanding of how and where BGCs are maintained is essential. To determine patterns of clonality and defensive BGC gene gain/loss on local scales, whole-genome sequencing was completed for 13 Pseudonocardia symbionts that were isolated from a single ant colony. Symbiont chromosomes from the same T. septentrionalis colony were clonally related, with very few SNPs or regions of recombination separating strains and limited gene content differences between them. However, plasmid SNPs and gene content are much more variable between strains, and plasmid-borne BGCs can be strain specific. These results suggest that plasmids may be rich sites for accruing BGC diversity that can be targeted for drug discovery.

More broadly, we isolated Pseudonocardia symbionts from colonies from across the Eastern USA to test the effect of the ant host’s geographic distribution on the population structure of Pseudonocardia. Although Pseudonocardia symbiont diversity did not correlate with host biogeography, BGC diversity is conserved locally. Many of the BGCs identified in this study did not match known compounds, and may be novel. Geographic regions with unique BGC compositions can therefore potentially be targeted for antimicrobial discovery. This thesis presents a framework for contiguously and accurately sequencing Pseudonocardia symbiont genomes for drug discovery, and showed that BGC diversification is a source of local adaptation in an otherwise clonal background.

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