Studies in directed endosymbiosis

Cournoyer, Jason

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https://hdl.handle.net/2142/124413 Copy

Description

Title

Studies in directed endosymbiosis

Author(s)

Cournoyer, Jason

Issue Date

2024-04-26

Director of Research (if dissertation) or Advisor (if thesis)

Mehta, Angad

Doctoral Committee Chair(s)

Mehta, Angad

Committee Member(s)

• Sarlah, David
• Hergenrother, Paul
• Luthey-Schulten, Zaida

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

cyanobacteria, endosymbiosis

Abstract

The acquisition of membrane-bound organelles such as mitochondria and chloroplasts was a fundamental touchstone in eukaryotic cell evolution, and it has consequently shaped the course of evolution and ecology. With the advent of sequencing technology, there is now overwhelming evidence to support a once-controversial hypothesis: that mitochondria and chloroplasts evolved from previously free-living prokaryotes that were engulfed by larger cells and retained as endosymbionts. Over time, these endosymbionts ceded most of their own genomes, thereby making essential processes such as DNA replication dependent on peripheral factors: in eukaryotic organisms, essential hostencoded proteins must first localize to the organelle, meaning it is only within the host cell microenvironment that the organelle can persist. The survival of extant host cells, in turn, depends on central chemical processes carried out in the organelles which serve bioenergetic and catabolic functions, such as photosynthesis, the Calvin–Benson–Bassham (CBB) cycle, and the TCA cycle. Importantly, these processes are only known to have ever evolved in prokaryotes and their organellar descendants. Endosymbiosis throughout nature is marked by several hallmarks: genome minimization, acquisition of protein import systems, metabolic interdependency, loss of peptidoglycan, and replication control. In principle, synthetic systems designed to mimic these hallmarks can serve to provide crucial insights into the process through which free-living bacteria transitioned into organelles, and test models derived from phylogenetic inferences. The first chapter of this dissertation is an introduction which briefly summarizes our current theory of eukaryotic cell evolution through endosymbiosis. The introduction also reviews advances made in engineering the two most important organisms in this dissertation, Saccharomyces cerevisiae and Synechococcus elongatus PCC7942 (hereafter Syn7942): these studies serve as precedence for the experimental approaches used in each of the subsequent chapters. The second chapter of this dissertation is a reprint of a paper published in Nature Communications, which describes the first steps in engineering artificial, photosynthetic endosymbiosis using S. cerevisiae and Syn7942. In this system, Syn7942 cells are fused with mutant S. cerevisiae cells which are incapable of respiration; the cyanobacterial cells support the host bioenergetic functions by producing ATP through photosynthesis and exporting that ATP into the yeast from within. The yeast-cyanobacteria chimeras are characterized by analysis of their genomes and microscopy. The third chapter of this dissertation is a paper in preparation, detailing a study in which a variety of auxotrophic Syn7942 mutants were fused to yeast cells in order to demonstrate metabolic coupling between the host and endosymbiont. This study aims to mimic the loss of essential genes in endosymbiotic cyanobacteria during chloroplast evolution by iteratively deleting genes in Syn7942 which encode enzymes catalyzing steps in amino acid and coenzyme biosynthesis. In this system, the viability of these auxotrophic cyanobacteria as endosymbionts is measured in order to elucidate the need for acquisition of exogenous transport mechanisms. The fourth chapter of this study aims to use in situ hybridization analysis as a method to visualize how gene expression is altered in yeastcyanobacteria chimeras. Using this method, established protocols can be used to perturb the abundance of transcripts corresponding to metabolic proteins in either the host or endosymbiont. On a cell-by-cell basis, adaptations on the transcriptomic level which may affect the viability of chimeras can be observed. This study could provide insights into to how to modulate environmental conditions (e.g., light) to favor sustained endosymbiosis using the engineered organisms.

Graduation Semester

2024-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2024 Jason Cournoyer

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