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Has files: YesSubject: search.filters.subject.310504 Epigenetics (incl. genome methylation and epigenomics)

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    Characterisation of epigenomic variation in natural isolates of E. coli : a thesis submitted in partial fulfilment of the requirements for the degree of Ph.D in Genetics, Massey University, College of Science, School of Natural Sciences, Auckland
    (Massey University, 2023) Breckell, Georgia
    DNA methylation is ubiquitous in bacteria and has a range of roles including self versus non-self recognition, DNA repair, and regulation of gene expression in response to internal and external cues. Regulation of gene expression by DNA methylation can lead to the establishment of phenotypic variation in otherwise isogenic populations. Until recently methods for the genome-wide study of DNA methylation in bacteria have been limited and therefore the full extent of DNA methylation's role in bacterial genomes is not well understood. In this thesis I use Oxford Nanopore Technologies sequencing to investigate the presence and activity of DNA methyltransferase in natural isolates of E. coli. The first aim of this thesis is to produce high quality genome assemblies that can be used to determine methylation patterns. To achieve this, in Chapter 2 I first use in silico methods to quantify the effects of different read length characteristics on assembly quality. I then optimise DNA isolation and library prep methods to obtain high quality DNA. In Chapter 3 I apply the results of Chapter 2 to sequence 49 natural isolates of E. coli from across the E. coli clade. I next benchmark five genome assembly methods for assembly accuracy. I base accuracy on five metrics designed to measure both the overall structural accuracy and the sequence accuracy of each assembly. The large number of isolates (49) used in this study, allows identification of the strengths associated with each assembly method. These results quantitatively describe best practices for bacterial genome assembly and highlight the current variability in genome assembly accuracy and therefore the importance of tailoring assembly methods to the study objectives. Finally, in chapter 4 I use the data produced in Chapter 3 to investigate DNA methylation in three E. coli natural isolates. After in silico identification of all the methyltransferases in each genome, I show that the activity of all predicted methyltransferases can be detected, as well as the activity of unexpected putative methyltransferases which are present in our isolates. Finally, I show that the genome wide DNA methylation patterns show consistent differences across growth conditions. These results suggest that E. coli exhibits transient DNA methylation patterns depending on growth environment and state. Overall this thesis establishes methods for assessing genome assemblies and broadens our understanding of genome wide DNA methylation patterns and the dynamics of these patterns in E. coli. Additionally this work provides insight into the possibility of transient epigenetic differentiation in E. coli which is reflected in the DNA methylation patterns across the genome.
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    Rapid phenotypic switching in a natural isolate of Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand
    (Massey University, 2022) Pearless, Stella Margaret
    The survivability of any given bacterial population is dependent on its genetic and phenotypic makeup. When cells replicate they usually produce genetically identical daughter cells through a process called binary fission. Although these daughter cells may remain genetically identical and form isogenic populations, bacteria also possess the ability to alter their phenotype independently of other cells in their population. This can result in subpopulations of phenotypically variable cells forming within a larger population, rendering them phenotypically heterogeneous. Phenotypic heterogeneity can arise from multiple molecular mechanisms that cause either genetic or epigenetic changes. Genetic mechanisms include site-specific DNA inversion, slipped-strand mispairing and homologous recombination. Epigenetic changes involve modifications to DNA at the structural level, and in bacteria most commonly refer to methylation. Heterogeneity can provide evolutionary advantages through processes like bet-hedging or the division of labour. A well documented example of evolutionarily advantageous phenotypic heterogeneity is the formation of persister cells within bacterial strains, a leading cause of antibiotic treatment failure. In this study, we have identified an Escherichia coli natural isolate strain (SC375) that is able to rapidly switch between two phenotypes. The phenotypic heterogeneity demonstrated in this strain results in varying colony morphologies influenced by their cellular composition. We initially proposed a DNA inversion mechanism for this switching but subsequently confirmed that all cells remain isogenic regardless of cell phenotype. Through RNA-sequencing we identified three virulence genes that were differentially regulated in both phenotypes, suggestive of an epigenetic regulatory mechanism. We then show, using reporter assays, that two of these genes are expressed in variable levels across subpopulations. We suggest that the rapid phenotypic reversibility of this strain is a possible indicator of epigenetic memory.