Molecular mechanisms governing regulation of toxin-antitoxin systems in bacteria Open Access

Pavelich, Ian (Fall 2021)

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Bacteria utilize a vast array of systems to control their growth and metabolism. Tightly regulating the process of DNA replication, transcription, and/or translation significantly alters the fate of the cell, especially in response to stress. Modules termed “toxin-antitoxin systems” are encoded in all bacteria and impact essential cellular processes upon activation. Toxins, which are the effector molecules of these systems, are unique in that they are intracellularly beneficial for survival. They are not excreted to kill neighboring bacteria. Instead, their interaction with downstream targets impacts cellular metabolism as to aid bacteria in surviving a notably variable panel of stresses. In this dissertation, the molecular mechanisms governing the regulation of toxin-antitoxin systems are investigated by applying structural and biochemical approaches to a pair of distinct, well-characterized systems: Escherichia coli yefMyoeB and Proteus vulgaris higBhigA. First, prior studies of the YoeB toxin of yefMyoeB reveal that YoeB adopts a novel dimeric conformation in contrast to other toxins that are monomeric. Dimeric YoeB is just as active as an engineered monomeric YoeB variant. However, dimeric YoeB is more thermostable. This is important as YoeB is activated during heat stress in which adopting a more thermostable form is advantageous. Second, studies of higBhigA reveal that higBhigA is not regulated by any classically established method of autoregulation in toxin-antitoxin systems. Typically, regulation of toxin-antitoxin complexes is via a negative feedback loop and is sensitive to changing levels of toxin, which affect the ability of these complexes to bind and repress further activity from their own operon. In contrast to this, higBhigA appears to be insensitive to changing levels of toxin HigB. Together, these studies reveal novel insights into how toxins can target protein synthesis, and the role they play in self-regulation. As toxins can drastically affect available cellular building blocks and metabolism, it is important to understand their regulation in mechanistic detail. The knowledge of how YoeB utilizes a dimeric form to tolerate heat stress, or how higBhigA is regulated without the use of excess toxin, provides additional insight into the numerous mechanisms bacteria utilize to evade environmental stress. 

Table of Contents

Chapter 1 – Introduction to toxin–antitoxin systems in bacteria 1

1.1 Abstract 1

1.2 Toxin-antitoxin systems in bacteria 2

1.3 Regulation of toxin-antitoxin activity 4

1.4 Diversity of type II antitoxins 8

1.5 Diversity of type II toxins and targets 10

1.6 Activation of toxin-antitoxin systems 12

1.7 Questions addressed 15

1.8 Figures & Tables 19

1.9 References 24

Chapter 2 – Monomeric YoeB toxin retains RNase activity but adopts an obligate dimeric form for thermal stability 43

2.1 Abstract 44

2.2 Introduction 45

2.3 Materials and Methods 49

2.3a Strains and plasmids 49

2.3b Purification of YefM, YoeB, and YoeB variants 49

2.3c In vitro mRNA cleavage assays 51

2.3d Thermus thermophilus 30S complex formation and crystallization 51

2.3e Thermus thermophilus 70S complex formation, crystallization, and structure determination 52

2.3f Site-directed mutagenesis 53

2.3g Differential scanning fluorimetry 54

2.4 Results 55

2.4a YoeB cleaves mRNA bound to both the 30S subunit and the 70S 55

2.4b Molecular recognition of the 30S subunit by the YoeB dimer 56

2.4c Molecular mechanism of YoeB recognition of a UAA stop codon 58

2.4d Molecular mechanism of YoeB recognition of a AAU asparagine sense codon 61

2.4e YoeB-induced conformational changes of the decoding center 62

2.4f Monomeric YoeB retains ribosome-dependent ribonuclease activity yet is thermally unstable 63

2.5 Discussion 65

2.6 Acknowledgements 71

2.7 Funding 72

2.8 Figures & Tables 73

2.9 References 98

Chapter 3 – Transcriptional repression of the hig toxin-antitoxin locus is independent of HigBHigA toxin-antitoxin oligomeric state 108

3.1 Abstract 109

3.2 Introduction 110

3.3 Materials & Methods 114

3.3a Strains and plasmids 114

3.3b HigA, HigB, and HigBHigA expression and purification 114

3.3c Crystallization, data collection and structure determination of HigBHigA-O2 DNA complexes 114

3.3d Molecular dynamics simulations 116

3.3e Electrophoretic mobility shift assays (EMSAs) 117

3.3f Dianthus binding assays 118

3.3g Differential scanning fluorimetry (DSF) 118

3.3h β-galactosidase assays 118

3.3i Western blot analysis 119

3.4 Results 121

3.4a Structure of HigB2HigA2-O2 DNA 121

3.4b Structure of HigBHigA2-O2 DNA 123

3.4c HigB2HigA2-O2 and HigBHigA2-O2 complexes exhibit similar dynamics 124

3.4d HigBHigA binding at O1 or O2 operators is independently regulated 125

3.4e Both trimeric HigBHigA2 and tetrameric HigB2HigA2 repress Phig 127

3.5 Discussion 130

3.6 Acknowledgements 134

3.7 Funding 135

3.8 Figures & Tables 136

3.9 References 162

Chapter 4 – Stability of toxin-antitoxin complexes governed by regulated antitoxin proteolysis 170

4.1 Abstract 171

4.2 Introduction 172

4.3 Materials & Methods 176

4.3a Strains and plasmids 176

4.3b Bacterial growth and cell viability assays 176

4.3c YefM and YoeB expression and purification 177

4.3d Western blotting analysis (DinJ, RelB, and YefM) 177

4.3e Western blotting analysis (FLAG) 178

4.3f α-DinJ ELISA 179

4.4 Results 181

4.4a C termini of DinJ and RelB, but not YefM, are essential for toxin inhibition 181

4.4b 3xFLAG DinJ is artificially stabilizing 183

4.4c Validation of anti-RelB and anti-YefM antibodies for future use 184

4.4d Establishing an anti-DinJ ELISA for high-throughput screening of antitoxin half-life 185

4.5 Discussion 186

4.6 Figures & Tables 190

4.7 References 204

Chapter 5 – Conclusion 211

5.1 Abstract 211

5.2 Introduction 212

5.3 Toxin activity as a consequence of stress 214

5.4 Type II toxin-antitoxin systems are regulated by a diverse set of mechanisms 219

5.5 Toxin activation by antitoxin proteolysis remains poorly understood 220

5.6 Concluding Remarks 222

5.7 Figures 224

5.8 References 229

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