Regulation of Gene Expression by DNA Methylation Patterns in E. coli

Phase variation is a form of heritable, but potentially reversible gene regulation, which occurs in bacteria. One of the regulatory mechanisms requires the heritable DNA methylation state of specific sites, as has been elucidated for phase variation of a group of fimbrial operons in Escherichia coli. This methylation-dependent regulation requires deoxyadenosine methylase (Dam). The biological significance of specifically Dam-dependent phase variation and how the DNA methylation state is inherited is yet to be understood. This research aims to address these questions by studying the molecular mechanism of Dam-dependent phase variation of the non-fimbrial outer membrane protein Ag43 in E. coli, and by testing the hypothesis that phase variation is important for Ag43-dependent biofilm formation. The long-term goal is to understand the biological significance and mechanisms of heritable gene regulation in bacteria, specifically those involving DNA modification. Dam-dependent regulation of Ag43 requires the oxidative stress response regulatory protein OxyR. OxyR is proposed to repress Ag43 expression by binding to the regulatory region of the Ag43 encoding gene, which contains Dam target sites. Transcription is thought to occur when OxyR binding, and thus repression, is abrogated by methylation of those Dam target sites. In Aim 1 the specific roles of Dam and OxyR in transcription and phase variation will be addressed. In vitro and in vivo analyses will be used to analyze transcription, and genetic approaches will be used to identify essential regulatory elements. The Ag43 locus has been implicated in affecting biofilm formation. Biofilms may contribute to bacterial survival and persistence in the environment. In Ag43 mutants, biofilm formation is defective under certain growth conditions. However, the effect of phase variation of Ag43 on this tractable and biologically relevant process,has not been addressed. Aim 2 will define the role of Ag43 phase variation in biofilm formation using isolates with well-defined mutations in Ag43 expression. In addition, it will be determined whether Ag43 regulation is altered during growth in a biofilm, addressing the adaptive potential of the regulatory mechanism. Regulation of Ag43 expression is an amenable system to address questions concerning the heritable nature of Dam-dependent gene regulation. In Aim 3, cellular or physiological factors that are required to maintain the DNA methylation and gene expression state will be identified by isolating and characterizing mutants with altered switch frequencies. DNA replication can result in a change in DNA methylation state. Therefore, the role of DNA replication in initiating the switch in transcription phase will also be specifically addressed. Various bacterial species contain Dam homologues, suggesting that this mechanism of gene regulation is widespread. Supporting this hypothesis and the importance of this modification system was recently underscored by the observation that Dam is required for Salmonella virulence. Additionally, DNA methylases that do not seem to be part of a restriction-modification system are also found in other species, where they may be involved in gene regulation. Principles elucidated in this study may therefore be applicable to various bacterial species. Heritable gene regulation like phase variation, dictates gene expression in a daughter cell, and thus affects the composition of a bacterial population over more than one generation. Therefore examining the significance of phase variation will increase our understanding of the behavior of bacterial populations. Finally, by addressing how DNA methylation patterns are formed and maintained in a bacteria, general underlying principles can be elucidated that may be applicable to more complex methylation-dependent regulatory systems, perhaps even those in eukaryotic organisms.