Date Thesis Awarded
5-2024
Access Type
Honors Thesis -- Access Restricted On-Campus Only
Degree Name
Bachelors of Science (BS)
Department
Biology
Advisor
Mark Forsyth
Committee Members
Kurt Williamson
Bjorg Larson
Abstract
Beyond the medical relevance of the gastric pathogen Helicobacter pylori, this gram-negative bacterium is an important model of bacterial transcriptional regulation due to its small genome size. This study aimed to further elucidate how phase variation, DNA methylation, and regulatory RNAs play a role in H. pylori gene expression control. We demonstrated that phase variation in the restriction endonuclease gene hsdR1 controls expression of the downstream DNA methyltransferase hsdM1 encoded in the same operon. To investigate if regulation of this methyltransferase controls DNA-methylation-dependent changes in gene expression, our lab identified DNA methylation sites in H. pylori strain 26695 using a hsdM1 deletion mutant to determine the DNA motif methylated by this enzyme. Traditional epigenetic theory holds that methyl groups physically block RNA polymerase from accessing a promoter to reduce transcription. However, we showed that transcript levels of putative genes methylated by HsdM1 do not change in hsdR1 phase variation mutants, suggesting there may be additional complexity to this system. Our methylome determination also revealed a 5’-RCGDAD-3’ motif methylated at a high level throughout the H. pylori genome by an unknown methyltransferase. Two of these methylated motifs are shortly after the transcription start site for a gene encoding the regulatory RNA HPnc4160, which regulates H. pylori virulence factors including cytotoxin-associated gene A (CagA). We demonstrated that there is a site-dependent effect of DNA methylation on expression of HPnc4160. As expected, DNA methylation of HPnc4160 does not affect cagA mRNA expression, but its effect on CagA protein expression is unclear. However, there is no effect of DNA methylation of HPnc4160 on CagA translocation or interleukin-8 production. Overall, this work highlights the nuance of bacterial gene expression control and delves into the role of DNA methylation and transcriptional pausing in the formation of stable regulatory RNAs.
Recommended Citation
Phillips, Celeste, "DNA Methylation and the Regulatory RNA HPnc4160 in Helicobacter pylori" (2024). Undergraduate Honors Theses. William & Mary. Paper 2145.
https://scholarworks.wm.edu/honorstheses/2145
