Mechanisms of DNA Modification-Dependent Regulation in Gram-Positive Bacteria
Nye, Taylor
2020
Abstract
The presence of DNA modifications is pervasive among both prokaryotic and eukaryotic species. In bacteria, the study of DNA methylation has largely been in the context of restriction-modification systems, where DNA methylation serves to safeguard the chromosome against restriction endonucleases that are intended to cleave invading foreign DNA. There has been a growing recognition that the methyltransferase component of restriction-modification systems can also function in the regulation of gene expression. Outside of restriction modification systems, DNA methylation from orphan methyltransferases, which lack cognate restriction endonucleases, have been shown to regulate critical cellular processes. The majority of research articles focuses on the epigenetic regulatory roles of bacterial DNA methylation in the context of Gram-negative bacteria, with particular bias towards Escherichia coli, Caulobacter crescentus, and related Proteobacteria. Despite the critical functions of DNA methylation in Gram-negative bacteria, far less is known about how DNA methylation contributes to epigenetic regulation of gene expression in Gram-positive bacteria. In this thesis I investigated the effects of DNA modifications in Gram-positive bacteria. I showed that DNA methylation from an active Type I restriction-modification system in Streptococcus pyogenes also functions in the epigenetic regulation of a small subset of virulence genes, all of which are significantly down regulated in the absence of DNA methylation. Moreover, I showed that the methylation-dependent decrease in gene expression results in attenuated virulence of an S. pyogenes clinical isolate, implicating DNA methylation as an important contributor to S. pyogenes pathogenesis. I also characterized the methylomes for two strains of the Gram-positive Firmicute Bacillus subtilis and demonstrated that DNA methylation regulates the expression of a small subset of genes involved in chromosome structure and maintenance. I further identified a methylation-sensitive transcriptional regulator, providing some of the first insight into the mechanisms of methylation-dependent gene regulation in Gram-positive bacteria. Finally, I identified a previously uncharacterized gene, rnhP, which is a plasmid encoded RNase HI. I found that RnhP contributes to genome maintenance in B. subtilis NCIB 3610 by removing RNA-DNA hybrids with four or more ribonucleotides embedded in DNA. I showed that RnhP does not contribute to plasmid maintenance or hyper-replication. Importantly, I showed that RnhP contributes to genome maintenance by allowing DNA replication forks to progress through the terminus region. Together, my work highlights the importance of DNA modifications and noncanonical nucleotides in Gram-positive bacteria and provides a framework for future studies of epigenetic regulation by RM systems in bacterial pathogenesis and development.Subjects
DNA methylation-dependent gene regulation in bacteria Ribonucleotide repair in bacteria
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