DISCOVERY OF THE ParST TOXIN-ANTITOXIN SYSTEM AND ITS PUTATIVE ROLE IN DISRUPTING NUCLEOTIDE METABOLISM

Abstract

Toxin-antitoxin (TA) systems are two-component operons that control cell growth, bacteriostasis, and death and have been implicated in important clinical issues such as biofilm formation and persister growth. Chapter 2 describes the extensive structural and biochemical elucidation of a TA system first predicted bioinformatically in 2009, here named ParST. We showed that this TA family is exceptionally widespread among bacteria, and a high-resolution crystal structure of the ParT toxin revealed similarity to known mono-ADP-ribosyltransferases, such as diphtheria and cholera toxins. Further experiments confirmed this function and identified phosphoribosyl pyrophosphate synthetase (Prs) as a putative target. Prs is an essential metabolic enzyme in nucleotide synthesis, not previously known to be targeted by cellular toxins, which makes ParST an interesting candidate for further study.

While Chapter 2 represents a new area of study in the lab, Chapters 3 through 5 return to the lab’s primary research interest – lasso peptides. Lasso peptides are ribosomally synthesized, post-translationally modified natural products that consist of an 7-9 amino acid ring, with the C-terminal portion of the peptide threaded through. In Chapter 3, for the first time, various noncanonical amino acids (ncAAs) were successfully introduced into lasso peptide microcin J25 (MccJ25). All 16 ncAA-substituted variants we examined were produced and retained antimicrobial activity against Salmonella newport. Work in this chapter has laid the groundwork for applications currently underway probing the interaction of MccJ25 with its target, RNA polymerase. Chapter 4 investigates the dynamics of lasso peptide isopeptidases, enzymes that cleave the characteristic lasso peptide isopeptide bond. We describe the creation of a dual-labeled isopeptidase capable of intramolecular fluorescence resonance energy transfer (FRET) for examining conformational changes, as well as preliminary results of FRET monitoring. Finally, Chapter 5 discusses the Sphingopyxis fribergensis family of lasso peptides, bergenodins 1 through 6. To date, six lasso peptides represents the most produced by a single organism. We confirm the production of bergenodins 1 through 5, though we could not isolate bergenodin 6. We also desired to examine any crosstalk between the many isopeptidase enzymes and lasso peptide substrates, but further optimization to the isopeptidase expression systems remains necessary.