Context is King: Understanding the Diversity and Evolution of Prokaryotic Metabolism through Comparative Contextual Methods

Author: Rusley, Calvin James

Year: 2027

Degree: Dissertation (Ph.D.)

Advisors: Orphan, Victoria J.; Fischer, Woodward W.

Committee Members: Leadbetter, Jared R.; Karthikeyan, Smruthi; Orphan, Victoria J.; Fischer, Woodward W.

Option: Geobiology

Abstract

Understanding the evolution and diversity of prokaryotic metabolism is highly dependent on protein annotation. Unfortunately, over the past decade, the rapid expansion of sequencing data has dramatically outpaced the ability of protein databases and bioinformatic tools to keep up, creating blind spots in standard annotation pipelines. By leveraging contextual evidence from protein phylogeny and predicted structure, genomic collocation, environmental and ecological settings, and inter-amino acid associations within proteins, this thesis seeks to plug these blinds spots in order to explore the metabolisms of uncharacterized prokaryotic clades. In the Gemmatimonadota, I find that members of the phylum encode a panoply of redox-active protein complexes, including heretofore unrecognized, vertically inherited Alternative Complex III. Next, I examine the global distribution and genomic potential of the understudied marine sedimentary phylum Aerophobota, including an apparent proclivity for adding a specific redox domain to its protein complexes. Elsewhere in marine sediment, I investigate the metabolism of anaerobic methanotrophic archaea in the absence of their traditional bacterial partners. Lastly, I present PEAPOD, a modular python package that uses Protein Language Models to align extremely evolutionarily divergent proteins, like those of the Phototrophic Reaction Center superfamily. Together, these chapters serve as a model for investigating prokaryotic metabolism amid an overwhelming, but useful, mountain of sequencing data.