Synthetic and systems biology approaches to characterizing the biochemical events underlying gyrase inhibitor-induced cell death in Escherichia coli.

摘要

The elucidation and characterization of cellular gene expression and phenotypic responses from the structure of gene regulatory networks is among the focal points of systems biology. Along these lines, one objective of the emergent field of synthetic biology, in which engineered genetic circuits are interfaced with the natural regulatory architecture of the cell, is to expand our knowledge of natural biological networks. As such, the employment of synthetic experimental platforms, and thus "programmable cells", in conjunction with systems-level analyses presents one with a powerful methodology for use in studies aimed at providing new biological insight. Accordingly, this thesis explores the intersection of synthetic and systems biology and culminates in the discovery of a novel, DNA gyrase inhibitor-induced oxidative damage cellular death pathway. We first designed an artificial, RNA-based gene expression system for precise regulation of mRNA translation in prokaryotes (referred to as riboregulation). This system exploits RNA's structure forming potential for tight cis-repression and sequence-specific binding capability for exacting trans-activation of protein expression. Next, we applied our riboregulation platform to the study of the endogenous gyrase inhibitor, CcdB. In-depth, in vivo characterization of this peptide poison, which typically ensures proper segregation of the F plasmid, has been prohibited by its potent toxicity and available experimental techniques. Using our system, we were able to phenotypically monitor the effect of CcdB in several E. coli strains. In addition, we profiled the gene expression response of these strains to CcdB poisoning and taking a systems biology approach in our analysis, identified several unique and significantly changing biochemical pathways. Finally, we expanded our analysis of gyrase inhibition to include functionally analogous quinolone antibiotics, and attempted to uncover the series of secondary biochemical events which commonly contribute to cell death following gyrase poisoning. We demonstrate that gyrase inhibition induces the generation of superoxide and hydroxyl radical oxidative species, which are integral to cell killing. We also show that iron-sulfur clusters play a key role in this process---superoxide-based oxidation of iron-sulfur clusters promotes a breakdown in intracellular iron regulatory dynamics which, in turn, drives production of highly deleterious hydroxyl radicals.