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Uncovering the Mechanism of Action of Bacterial Vap, Doc, and Rel Toxins

by
Jonathan William Cruz
B.S., Rutgers University - 2007

Thesis Advisors: Nancy A. Woychik, Ph.D.
Graduate Program in Microbiology & Molecular Genetics

RWJMS Research/School of Public Health Building
Conference Room 258, Piscataway

Monday, August 11, 2014
1:00 pm


Abstract

Bacteria have exquisitely adapted to life in extreme environments, possibly due to the evolution of mechanisms allowing them to resist and tolerate various stresses. Recent studies have forged a strong link between the abundance of toxin-antitoxin (TA) systems in a bacterial genome and stress survival. TA systems are autoregulated two gene operons encoding a stable toxin, which targets essential cellular processes, along with a labile antitoxin that binds to and neutralizes its cognate toxin. Though prevalent in bacteria, the mechanism of action of many TA system toxins remains poorly understood. We investigated the biochemical effects of three TA toxins: VapC, Doc, and RelE. First we studied VapC-mt4 from Mycobacterium tuberculosis as a representative of this class of toxins. We found that expression of VapC-mt4 inhibits growth and arrests translation. However unlike many other TA toxins that affect protein synthesis, VapC-mt4 targets a specific subset of tRNAs for cleavage at a single site in their anticodon loops. The second toxin we studied was the Doc protein from bacteriophage P1. Doc belongs to the Fic family of proteins whose members typically inactivate targets via adenylylation. We determined that Doc inhibits translation in Escherichia coli by modifying the essential GTPase EF-Tu. In contrast to other Fic proteins, Doc is a kinase that phosphorylates a conserved threonine residue at the C-terminus of EF-Tu. The final TA toxin we studied was RelE from E. coli. Contrary to earlier in vitro experiments suggesting a preference for cleavage at stop codons, our in vivo results revealed that RelE extensively cleaves mRNA at the 5 end of the open reading frame in a codon-independent manner. Overall we have discovered several novel mechanisms of action for TA toxins. Each of these toxins inhibit translation, but they do so by targeting very different components of this important cellular process: tRNAs, elongation factors, or mRNA. Consequently, each toxin alters translation in a distinct manner and to varying degrees, which may account for the variability in how bacteria differentially fine tune their response to stress in order to maximize their survival.


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