Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase IV Activities
DNA gyrase and DNA topoisomerase (topo) IV are the bacterial targets of coumarin and quinolone antimicrobial agents. Widespread resistance to clinically important antibiotics such as beta-lactams and macrolides has stimulated the development of novel gyrase and topo IV inhibitors especially against Streptococcus pneumoniae and other Gram-positive pathogens. Here, we describe how gyrase and topo IV activities are measured and how inhibitors of these enzymes may be assayed, focusing as a paradigm on DNA supercoiling by S. pneumoniae gyrase, DNA decatenation by S. pneumoniae topo IV, and DNA cleavage by both enzymes. These approaches provide mechanistic insight on inhibitor action and allow identification of dual gyrase/topo IV targeting agents that can minimize the emergence of bacterial resistance.
Key WordsDNA gyrase DNA topoisomerase IV DNA supercoiling DNA decatenation cleavage complex ciprofloxacin agarose gel electrophoresis Streptococcus pneumoniae
- 5.Aubry, A., Veziris, N., Cambau, E., Truffot-Pernot, C., Jarlier, V., and Fisher, L. M. (2006) Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis: Functional analysis of mutant enzymes. Antimicrob. Agents Chemother. 50, 104–112.CrossRefPubMedGoogle Scholar
- 13.Grossman, T. H., Bartels, D. J., Mullin, S., Gross, C. H., Parsons, J. D., Liao, Y., Grillot, A.L., Stamos, D., Olson, E. R., Charifson, P. S., and Mani, N. (2007) Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds. Antimicrob. Agents Chemother. 51, 657–666.CrossRefPubMedGoogle Scholar
- 18.Pan, X.-S., Yague, G., and Fisher, L. M. (2001) Quinolone resistance mutations in Streptococcus pneumoniae GyrA and ParC proteins: Mechanistic insights into quinolone action from enzymatic analysis, intracellular levels, and phenotypes of wild-type and mutant proteins. Antimicrob. Agents Chemother. 45, 3140–3147.CrossRefPubMedGoogle Scholar
- 19.Sayer, P. J., Goble, M. L., Oram, M., and Fisher, L. M. (2001) Plasmid supercoiling by DNA gyrase. In Methods in Molecular Biology, Vol. 95: DNA Topoisomerase Protocols, Part II: Enzymology and Drugs (N. Osheroff and M. A. Bjornsti, eds.), Humana Press Inc, Totowa, NJ, pp. 25–33.Google Scholar