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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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    Pangenome-guided tools for investigating the role of epsilonproteobacteria in human gastroenteritis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Science at Massey University, Manawatu, New Zealand
    (Massey University, 2018) Cornelius, Angela Joyce
    Gastroenteritis affects billions of people every year and current testing methods fail to identify the cause for approximately half of the samples submitted for microbiological testing. Epsilonproteobacteria contains Campylobacter jejuni, the most commonly reported cause of bacterial gastroenteritis in the world, and Helicobacter pylori, a gastric pathogen and class I carcinogen. This bacterial class also contains ≥20 additional species known, or suspected, of being human pathogens. To better understand the role some of these species play in human gastroenteritis, novel rapid, cost effective methods are needed. The growing number of whole genome sequences available for this class were exploited to first evaluate the classification of the genetically heterogeneous species C. concisus and then to identify taxon-specific CDS for a range of Epsilonproteobacterial taxa. Probes were designed to detect 28 of these CDS and incorporated into a single multiplex ligation-dependent probe amplification (MLPA) assay which was tested against DNA from 43 Epsilonproteobacterial species and then applied to DNA extracts from stool samples from a childhood gastroenteritis case control study undertaken in Belgium. The 22 C. concisus genomes consistently clustered into two genomospecies (GS) represented by ATCC 33237T (GS1) and CCUG 19995 (GS2). Taxon-specific genes were identified for 28 taxa, including the two C. concisus genomospecies, and concordant results were observed for the majority of MLPA probes and DNA extracts from pure cultures. The probes designed to detect C. lari subsp. concheus and H. pullorum failed to detect the target DNA; all of the urease positive thermophilic Campylobacter DNA extracts were also positive for the probe designed to detect C. subantarcticus, some probes lacked repeatability in the presence of elevated EDTA and the size differences between some probes needs to be optimised. C. jejuni was the most common Epsilonproteobacterial species isolated by culture and C. concisus was the most common species detected by MLPA. Both C. jejuni and C. concisus GS2 were detected in significantly higher numbers in cases than controls in a Belgian childhood case control study. This demonstrated the utility of the Epsilonproteobacteria MLPA assay and provides some evidence that C. concisus GS2 may have a role in childhood gastroenteritis.
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    The evolution of selfish genetic elements within bacterial genomes : a thesis submitted in partial fulfilment of the requirements for the degree of Ph.D. in Molecular Evolution at Massey University, Auckland, New Zealand
    (Massey University, 2012) Bertels, Frederic
    Genes that increase their copy number relative to that of the host genome are termed selfish. Selfish genes are found ubiquitously in bacterial genomes. Within genomes they can often be identified due to their repetitive nature. Short repetitive sequences such as repetitive extragenic palindromic (REP) sequences have been proposed to be selfish genetic elements. However, evidence for the selfishness of REPs is scarce due to the lack of knowledge about their origin, evolution and mechanisms of dispersal. Here, REPs are studied in the model bacterium Pseudomonas fluorescens SBW25. The evidence provided suggests that REPs are part of a greater mobile genetic element, which is termed REP doublet forming hairpins (REPINs). Subsequently, I investigate the cause of REPIN dispersal: a putative transposase. The transposase, named REP-associated tyrosine transposase (RAYT) shares essential motifs with the IS200 family of insertion sequences. However, unlike insertion sequences, RAYTs are found only as single copy genes. This indicates that RAYTs may not be entirely selfish; instead they may have been co-opted by the host to perform a beneficial function. Finally, two more repetitive sequence classes are studied in the SBW25 genome. Interestingly, both sequence classes consist of a protein coding sequence and a sequence that forms a stable secondary structure in single stranded DNA or RNA. This arrangement is reminiscent of bacterial toxin-antitoxin (TA) systems. Evidence from sequence analyses suggests that the repetitive nature of these elements in SBW25 may be the result of cooperation between REPINs or other replicative elements and the TA systems. The presented analyses show that despite the streamlined nature of bacterial genomes selfish genetic elements frequently arise, replicate and probably increase theirpersistence and spread through cooperation with addictive and duplicative elements respectively. persistence and spread through cooperation with addictive and duplicative elements respectively.