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Mechanisms of Resistance

Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

Xiao-Su Pan, L. Mark Fisher
Xiao-Su Pan
Molecular Genetics Group, Department of Biochemistry, St. George’s Hospital Medical School, University of London, London SW17 0RE, United Kingdom
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L. Mark Fisher
Molecular Genetics Group, Department of Biochemistry, St. George’s Hospital Medical School, University of London, London SW17 0RE, United Kingdom
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DOI: 10.1128/AAC.43.5.1129
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ABSTRACT

Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

  • Copyright © 1999 American Society for Microbiology
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Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones
Xiao-Su Pan, L. Mark Fisher
Antimicrobial Agents and Chemotherapy May 1999, 43 (5) 1129-1136; DOI: 10.1128/AAC.43.5.1129

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Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones
Xiao-Su Pan, L. Mark Fisher
Antimicrobial Agents and Chemotherapy May 1999, 43 (5) 1129-1136; DOI: 10.1128/AAC.43.5.1129
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KEYWORDS

anti-infective agents
DNA Topoisomerases, Type II
fluoroquinolones
Gene Expression Regulation, Bacterial
Streptococcus pneumoniae

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