Purification and Supercoiling of pBR322 DNA

Full-length wild-type S. aureus gyrase subunits (GyrA and GyrB, used for enzymological studies), as well as the wild-type gyrase core fusion truncate (GyrB27-A56) and a fusion truncate containing a GyrA^Y123F mutation (used for structural studies) were expressed and purified as described previously.^{25 (link)}Negatively supercoiled pBR322 DNA was prepared from Escherichia coli using a Plasmid Mega Kit (Qiagen) as described by the manufacturer. Positively supercoiled pBR322 DNA was prepared by treating negatively supercoiled molecules with recombinant Archaeoglobus fulgidus reverse gyrase.^{49 (link)–50 (link)} The number of positive supercoils induced by this process is comparable to the number of negative supercoils in the original pBR322 preparations.^{49 (link)} In the experiments that compared negatively and positively supercoiled DNA, the negatively supercoiled plasmid preparations were processed identically to the positively supercoiled molecules except that reaction mixtures did not contain reverse gyrase. Relaxed pBR322 plasmid DNA was generated by treating negatively supercoiled pBR322 with calf thymus topoisomerase I (Invitrogen) and purified as described previously.^{27 (link)}Gepotidacin was provided by GlaxoSmithKline. Moxifloxacin was obtained from LKT Laboratories. Gepotidacin and Moxifloxacin were stored at 4 °C as 20 mM stock solutions in 100% dimethyl sulfoxide.