Lethal Action of Quinolones against a Temperature-Sensitive dnaB Replication Mutant of Escherichia coli

Abstract

Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.Quinolones are broad-spectrum antibacterials that trap DNA gyrase and DNA topoisomerase IV on DNA as ternary complexes containing double-strand DNA breaks (reviewed in reference 5). The ternary complexes, which block DNA replication (8, 23), RNA synthesis (13, 24), and cell growth (7), lead to cell death by poorly understood processes. Quinolone-mediated inhibition of DNA replication, which is very rapid (19), is unlikely to account for rapid lethality because inhibition is reversible (4, 8) and because concurrent interruption of RNA or protein synthesis eliminates the lethal action of some quinolones but not their ability to block DNA replication (2, 4). A different issue is whether ongoing DNA replication is required for quinolone lethality. Such a hypothesis could be derived from studies with other topoisomerases in which collision of replication forks with ternary complexes is thought to release double-strand DNA breaks (3, 21). To address this possibility with quinolones, we blocked DNA replication by shifting a dnaB mutant of Escherichia coli to nonpermissive temperature. (DnaB is a DNA helicase [11] required for replication fork movement [1].) Subsequent treatment with quinolone killed the mutant, indicating that ongoing DNA replication is not required.E. coli strain K12SH-28 and its dnaB-22(Ts) mutant FA22 (6) were obtained from the E. coli Stock Center (Yale University) and were grown at 28°C in M9-glucose minimal medium (14) supplemented with 0.05% Casamino Acids and 1% LB medium (14). A gyrA quinolone-resistant mutant of FA22, strain KD2672, was constructed by P1-mediated transduction (22) from strain KD2346, a spontaneous ciprofloxacin-resistant derivative of KD1366 (26) that contained GyrA amino acid substitutions Ser-83 to Leu and Asp-87 to Tyr. The dnaB-22 allele was transduced into strain DM4100 (20) using a nearby tetracycline resistance marker (malF-3089:Tn10) to produce strain KD2773. Nalidixic acid, chloramphenicol, and tetracycline were products of Sigma Chemical Co. (St. Louis, MO); ciprofloxacin was obtained from Bayer Corp. (West Haven, CT). Bactericidal activity was determined by incubation in the presence of quinolone followed by dilution and growth on drug-free agar at 28°C for 1 to 2 days. The rate of DNA synthesis was measured by incubating 100 l bacterial culture with 0.1 Ci [ 3 H]thymidine for 2 min followed by determination of acid-precipitable radioactivity.When the dnaB-22 mutant was grown at 28°C and shifted to 42°C, the rate of DNA synthesis dropped by more than 95% within 10 min and was not inhibited further by subsequent nalidixic acid treatment (not shown). Under these conditions, the viable cell number r...

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