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Abstract

The evolution of bacterial resistance-to-phage strategies, and the mechanisms by which phages adapt to overcome these strategies, are usually studied via evolution experiments in which phages are added to sensitive bacteria and allowed to co-evolve. While this top-down approach has improved our understanding of co-evolutionary dynamics, it underestimates the diversity of available evolutionary pathways. In this thesis work, I propose an evolutionary bottom-up approach to study the natural lipopolysaccharide (LPS) structure diversity in resistant E. coli C strains and the corresponding ΦX174 adaptation process. I first investigated the mutational causes responsible for ΦX174-resistance using whole-genome sequencing. All phage-resistant mutants carry at least one mutation in genes linked to core LPS biosynthesis, assembly or regulation. Based on which genes are mutated and the current knowledge of LPS biology, I predicted that these bacterial strains collectively produce eight different LPS structures. Then, I used serial transfer experiments to evolve ΦX174 against each resistant mutant and determined the set of mutations required to overcome resistance. ΦX174 overcomes LPS-based resistance via mutations in only two genes: the F gene involved in host recognition and the H gene involved in DNA injection. Finally, I tested my predicted LPS genotype to phenotype maps using multiple phage infection assays to qualitatively determine the host range of each evolved phage. I showed that my evolved ΦX174 strains are great biosensor tools that can discriminate different E. coli C resistant strains. I also demonstrated that the LPS diversity is much greater than predicted. Instead of eight LPS structures, my phage analysis suggests that there are at least 14 distinct LPS phenotypes. My approach allows a deeper understanding of bacteria-phage interactions essential for developing efficient phage therapies in the future.