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Genetic bases and phenotypic consequences of high-temperature adaptation in Escherichia coli

  • Author(s): Rodriguez Verdugo, Alejandra
  • Advisor(s): Gaut, Brandon S
  • et al.
Abstract

Connecting phenotype to genotype and fitness has been a major challenge when studying adaptation. Experimental evolution is a powerful method to facilitate the study of adaptation as an outcome and as a process. This dissertation used 114 clones of Escherichia coli that were evolved independently for 2000 generations under thermal stress (42.2ºC). The goal of my dissertation was to link phenotypes and fitness with specific mutations and to identify the functional mechanisms leading to thermal adaptation.

Chapter 1 focused on a subset of populations that became resistant to an antibiotic, rifampicin, during thermal stress adaptation. Rifampicin resistance was caused by three mutations at codon position 572 of the rpoB gene, which encodes the beta subunit of RNA polymerase (RNAP). I used samples from 200 generation intervals to assess the frequency trajectory of rifampicin resistance. I found that resistant mutations typically appeared, and were fixed, early in the evolution experiment. Finally, I confirmed that the three mutations conferred high advantage in glucose-limited medium at 42ºC in the ancestral background.

Chapter 2 investigated the same rpoB mutations in codon 572 to explore the molecular mechanisms underlying their fitness improvement at high temperature. I measured their growth curves and gene expression (mRNAseq) at 42ºC, and compared them to the growth and gene expression of the ancestor at 37ºC and 42ºC. Two rpoB mutations restored the gene expression back to an ancestral state, while one mutation, exaggerated the expression changes of the ancestor at 42ºC. Lastly, I compared the phenotypic characteristics of one single mutant, I572L, to those of two high-temperature adapted clones with this mutation. I concluded that the I572L mutation contributed to most of the expression changes while later mutations did not substantially changed gene expression.

Chapter 3 explored the phenotypic consequences of high temperature adaptation in 114 clones. I measured the magnitude of fitness trade-offs across a thermal gradient. I identified two niche dynamics that I associated with two genetic pathways. Overall, my dissertation associates mutations to phenotypes in the context of thermal stress adaptation, and it highlights the difficulties of connecting genotype to phenotype, even if in a "simple" experimental system.

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