Chemical Approaches to Elucidate Novel Facets of Nucleic Acid Metabolism and Signaling Público
Fontaine, Benjamin (Fall 2018)
Abstract
Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms. Additionally, certain nucleotide signalling molecules, such as adenosine 3’,5’-cyclic monophosphate (3’,5’-cAMP) and bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP), regulate organismal adaptations to environmental stimuli. Recently, functions have emerged for certain non-canonical nucleotides, including cytidine 3’,5’-cyclic monophosphate (3’,5’-cCMP) and the regioisomeric nucleoside 2’,3’-cyclic monophosphates (2’,3’-cNMPs), but the underlying mechanisms remain largely elusive. Prior studies have suggested the existence of unique enzymes involved in 3’,5’-cCMP metabolism and binding; this work seeks to identify these potential enzymes through the development of affinity-based purification procedures and activity assays developed herein. In addition, LC-MS/MS methodology has been optimized to quantify 3’,5’- and 2’,3’-cNMP levels in various biological samples, resulting in the first quantification of 2’,3’-cNMP pools in Escherichia coli. These studies have revealed that 2’,3’-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, the present work reports the dissection of cellular processes modulated by RNase I and 2’,3’-cNMPs through the development of cell-permeable 2’,3’-cNMP analogs and a 2’,3’-cyclic nucleotide phosphodiesterase. Utilization of these (bio)chemical tools, in conjunction with phenotypic and transcriptomic investigations, has identified different pathways regulated by 2’,3’-cNMPs and RNase I, including chemotaxis, biofilm production, antibiotic tolerance, and nucleotide pool homeostasis. Along with chemical approaches to probe cryptic nucleotide-mediated signalling, novel chemical modulators of biofilm formation have been synthesized, providing additional tools to investigate microbial transduction. Through the development of these methods, this dissertation has elucidated enigmatic features of signal transduction, with a focus on understudied aspects of nucleotide metabolism.
Table of Contents
Chapter 1: Introduction to nucleotide signaling and metabolism
1.1 Nucleotide second messenger signaling………………………………………….2
1.2 Primary nucleotide metabolism in regulation of prokaryotic pathways………….8
1.3 Objectives and scope of this dissertation………………………………………...13
1.4 References………………………………………………………………………..15
Chapter 2: RNase I regulates Escherichia coli nucleoside 2’,3’-cyclic monophosphate levels and biofilm formation
2.1 Introduction……………………………………………………………………....21
2.1.1 Discovery of nucleoside 2’,3’-cyclic monophosphates and the early characterization of RNase I……21
2.1.2 RNA decay in bacteria…………………………………………………26
2.2 Results…………………………………………………………………………....30
2.3 Discussion………………………………………………………………………...39
2.4 Materials and Methods…………………………………………………………....43
2.5 References………………………………………………………………………...54
2.6 Supplementary Material…………………………………………………………..62
2.7 Supplementary References………………………………………………………..72
Chapter 3: Chemical tools reveal diverse functions modulated by RNase I and nucleoside 2’,3’-cyclic monophosphate pools in Escherichia coli
3.1 Introduction……………………………………………………………………….74
3.2 Results…………………………………………………………………………….77
3.3 Discussion………………………………………………………………………...88
3.4 Materials and Methods…………………………………………………………....96
3.5 References………………………………………………………………………..108
3.6 Supplementary Material……………………………………………………….....115
3.7 Supplementary References……………………………………………………….129
Chapter 4: Work toward elucidating the roles of cytidine 3’,5’-cyclic monophosphate in mammals
4.1 Introduction……………………………………………………………………....131
4.2 Results……………………………………………………………………………136
4.3 Discussion………………………………………………………………………...147
4.4 Materials and Methods…………………………………………………………....148
4.5 References………………………………………………………………………...158
Chapter 5: Development of phyto-inspired phenolic glycosides as inhibitors of Staphylococcus aureus biofilm formation
5.1 Introduction……………………………………………………………………….164
5.1.1 Antibiotic resistance and biofilm formation…………………………….164
5.1.2 Phenolic phytochemicals as modulators of bacterial biofilm productions and quorum sensing....165
5.2 Results……………………………………………………………………………. 169
5.3 Discussion……………………………………………………………………….. 175
5.4 Materials and Methods………………………………………………………..178
5.5 References…………………………………………………………………………191
5.6 Supplementary Material…………………………………………………………...196
Chapter 6: Conclusions, ongoing work, and future directions……………………………216
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