Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli

Daniel J Dwyer,Boris Hayete,Michael A Kohanski,James J Collins

doi:10.1038/msb4100135

Abstract

Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, double-stranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids.

Highlights

Bacterial chromosomal topology is maintained by the activities of topoisomerase I, topoisomerase IV and DNA gyrase (Champoux, 2001)
We detected a significant increase in hydroxyl radicalinduced HPF fluorescence at both 3 and 6 h post-treatment of wild-type E. coli with norfloxacin (Figure 3A)
We show that oxidative damage by superoxide and hydroxyl radicals contributes to bacterial cell death following gyrase poisoning

Summary

Introduction

Bacterial chromosomal topology is maintained by the activities of topoisomerase I, topoisomerase IV and DNA gyrase (topoisomerase II) (Champoux, 2001). The gyrase enzyme is responsible for the introduction of negative DNA supercoils in an ATP-dependent manner, and participates in the processes of replication, transcription, repair, recombination and decatenation (Gellert et al, 1976; Cozzarelli, 1980; Wang, 1996). Gyrase is composed of two subunits, gyrA and gyrB, and complexes with its DNA substrate as an A2B2 tetramer (Mizuuchi et al, 1978; Sugino et al, 1980). GyrA catalyzes the concomitant double-stranded breakage and rejoining of DNA phosphodiester bonds following binding and hydrolysis of ATP by GyrB (Gellert, 1981; Reece and Maxwell, 1991). The stable DNA–GyrA–inhibitor complex sterically inhibits replication and transcription by establishing a roadblock to DNA

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