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

Content Provider	PubMed Central
Author	Pan, Xiao-su Fisher, L. Mark
Copyright Year	1999
Abstract	Streptococcus pneumoniae gyrA and gyrB genes 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 against S. 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.
Starting Page	1129
File Format	PDF
ISSN	10986596
e-ISSN	10986596
Journal	Antimicrobial Agents and Chemotherapy
Issue Number	5
Volume Number	43
Language	English
Publisher	American Society for Microbiology
Publisher Date	1999-05-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Pharmacology Pharmacology (medical)

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Contribution of Topoisomerase IV and DNA Gyrase Mutations in Streptococcus pneumoniae to Resistance to Novel Fluoroquinolones

Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones.

Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

DNA Gyrase and Topoisomerase IV Are Dual Targets of Clinafloxacin Action in Streptococcus pneumoniae

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

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