Engineering Substrate Specificity and Investigating Regulatory Control of Bacterial Toxin-Antitoxin Systems Restricted; Files Only
Blackburn, Taylor (Summer 2026)
Abstract
Toxin-antitoxin (TA) systems are two-component genetic modules which encode for a toxic protein that targets a specific cellular process, and its cognate antitoxin or antidote which acts as a de facto control switch of toxin function. TA systems are ubiquitous among bacteria and have been attributed with functions in bacterial stress response, persistence, and phage defense, among other roles. These systems serve essentially as survival modules for the host cell that produces them, with their functions ultimately leading to slowed or halted cellular growth which enables survival of various environmental stressors. Toxins exhibit specificity for diverse targets including proteins, RNA, and DNA. For type II ribosome-dependent toxins which cleave mRNAs undergoing active translation on the ribosome, their specificity is exerted at the level of the mRNA codon sequence. Engineering the sequence specificity of these toxins may open the door to novel biotechnology tools and deepen our understanding of how toxins select for substrate. In Chapter 2, I determine that the substrate specificity of the RelE toxin can be engineered by manipulation of a residue near the catalytic site which does not directly participate in the catalytic mechanism of RNA cleavage. Type II TA systems are also known to self-regulate at the transcriptional level, exerting control over their own gene expression by binding upstream of the TA promoter. Transcriptional autoregulation is thought to depend on shifting ratios of toxin:antitoxin, but there remains little structural evidence of this phenomenon. We are starting to understand the diversity of interactions which TA systems employ to control transcription levels, and Chapter 3 provides biochemical and structural evidence of how the HigB toxin does not act as a traditional co-repressor of transcription but can still function as a de-repressor. Together, these studies provide novel insights into mechanisms of molecular recognition of type II TA systems.
Table of Contents
Chapter 1 ... 1
Introduction ... 1
1.1 Overview of Toxin-Antitoxin Systems ... 1
1.2 Prevalence of TA Systems in Microorganisms ... 2
1.3 Classification of TA Systems ... 3
1.4 Regulation of TA Systems ... 5
1.5 Cellular Roles of TA Systems ... 7
1.6 Engineering TA Systems ... 9
1.7 Contribution of this work to the TA field ... 12
1.8 Figures and tables ... 14
1.9 References ... 17
Chapter 2 ... 22
Engineering mRNA substrate specificity of the promiscuous E. coli type II toxin RelE ... 22
2.1 Abstract ... 23
2.2 Introduction ... 24
2.3 Materials and methods ... 30
2.4 Results ... 35
2.5 Discussion ... 47
2.6 Acknowledgements and funding ... 50
2.7 Figures and tables ... 51
2.8 References ... 66
Chapter 3 ... 70
Antitoxin control of optimal transcriptional repression in the atypical HigB-HigA toxin-antitoxin system from Proteus vulgaris ... 70
3.1 Abstract ... 72
3.2 Introduction ... 73
3.3 Materials and methods ... 76
3.4 Results ... 81
3.5 Discussion ... 91
3.6 Acknowledgements and funding ... 95
3.7 Figures and tables ... 96
3.8 References ... 120
Chapter 4 ... 124
Conclusions and Future Directions ... 124
4.1 Significance ... 124
4.2 Future exploration of TA regulation ... 125
4.3 Future exploration of toxin specificity ... 127
4.4 Concluding remarks and outlook ... 129
4.5 References ... 131
Appendix A ... 132
Developing a purification strategy for the Extraintestinal pathogenic E. coli PasT toxin and preliminary biochemical and structural studies ... 132
A.1 Abstract ... 133
A.2 Introduction ... 134
A.3 Materials and methods ... 136
A.4 Results ... 140
A.5 Discussion ... 146
A.6 Acknowledgements and funding ... 149
A.7 Figures and tables ... 150
A.8 References ... 160
Appendix B ... 162
An RNase toxin hijacks Elongation Factor-TU to cleave the ribosomal Sarcin-Ricin loop ... 162
B.1 Abstract ... 163
B.2 Introduction ... 164
B.3 Materials and methods ... 166
B.4 Results ... 178
B.5 Discussion ... 186
B.6 Acknowledgements and funding ... 191
B.7 Figures and tables ... 192
B.8 References ... 207
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File download under embargo until 20 August 2028 | 2026-08-11 17:25:36 -0400 | File download under embargo until 20 August 2028 |
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