title: DNA gyrase and DNA topoisomerase of Bacillus subtilis: expression and characterization of recombinant enzymes encoded by the gyrA, gyrB and parC, parE genes.
abstract: Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences. The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system. The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively. Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains. In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B. subtilis.
authors.affiliation: Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA. mbarnes@microbiotix.com
authors.forename: Marjorie H
authors.initials: MH
authors.lastname: Barnes
authors.forename: William A
authors.initials: WA
authors.lastname: LaMarr
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Foster
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004249:DNA Damage; D027081:DNA Gyrase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D027101:DNA Topoisomerase IV / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D024841:Fluoroquinolones / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D059005:Topoisomerase II Inhibitors
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D024841:Fluoroquinolones; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase