title: Phase variable methyltransferases associated with phasevarions
Creators: name: Scott, Adeana
Publisher: name: Griffith University
Year: 2021
Subjects: subject: DNA methyltransferases
subject: phasevarions
subject: Neisseria meningitidis

description: DNA methyltransferases catalyse the addition of a methyl group to recognition sequences within genomic DNA. Methylation of these sequences is a mechanism of self recognition utilised by multiple host adapted bacterial pathogens in restriction-modification systems. Additionally, multiple genes are up-regulated or down-regulated through methylation of overlapping DNA methyltransferase recognition sites and promoters. Identification of methyltransferase recognition sites is essential for investigation of the modulation mechanism of gene expression at the promoter level. Phase variation is the ON/OFF switching of gene expression resulting from changes in the number of short DNA repeats within an open reading frame or promoter region. ON/OFF switching of DNA methyltransferase expression epigenetically regulates gene expression in a phase variable manner. Phase variation of gene regulators such as DNA methyltransferases has been identified as a novel system known as phasevarions. Phasevarions are phase variable regulons. In this thesis the ModA11 DNA methyltransferase recognition sequence in Neisseria meningitidis was characterised. The random switching of ModA11 controls expression of over 80 genes. To further study of the ModA11 recognition sequence, a strain with ModA11 expression locked ON (modA11_1R) was generated to compare with locked OFF (modA11::kan). The ModA11 recognition sequence was identified as 5’-CGYm6AG-3’ through restriction inhibition assays and Single Molecule Real Time methylome analysis of modA11 ON, modA11_1R and modA11::kan knockout strains. The identification and confirmation of DNA methyltransferase recognition sequences were then able to facilitate the analysis of how changes in DNA methyltransferase expression regulate gene expression at the promoter level. In this study, firstly, the recognition sequences in the promoter regions of genes regulated in response to phase variable DNA methyltransferase expression described in an earlier publication were examined. The ON/OFF switching of ModA11 leads to the presence or absence of a N6-methyladenine modification at thousands of sites in the genome. Our bioinformatic analysis reveals only 9% of the regulated genes have a ModA11 methylation site located upstream with potential for direct effect of methylation on transcription. To further examine how phase variable DNA methyltransferase control of gene expression occurs at promoter level, a promoterless LacZ construct was generated to examine promoter sequences containing DNA methyltransferase target sites in the native strains. Using this lacZ gene fusion approach, this study showed that a single ModA11 methylation site upstream of the eda gene (Entner–Doudoroff aldolase) is sufficient to impart methylation-dependent regulation of Eda. Site-directed mutagenesis of the ModA11 site upstream of the Eda gene confirmed that methylation of this site modulates Eda expression. Eda is a component of the Enter- Doudoroff (ED) pathway which converts glucose to pyruvate and glyceraldehyde-3-phosphate. In N. meningitidis 69% of pyruvate is synthesized via the ED pathway. ModA11 phase variable methylation of the Eda regulon is therefore likely to trigger secondary affects affecting the Enter-Doudoroff pathway.