Purification of Citrobacter freundii DNA gyrase and inhibition by quinolones

Abstract

DNA gyrase is a bacterial enzyme which catalyzes the ATP-dependent negative supercoiling of DNA. It is the accepted target of quinolones. The enzyme from Citrobacter freundii IID976 was purified by affinity chromatography on novobiocin-Sepharose and heparin-Sepharose. It had two subunits, designated A and B, which closely resembled those of the enzyme from Escherichia coli and Micrococcus luteus in enzymatic requirements. The inhibitory effects of the quinolones on the supercoiling activities of the enzyme correlated with their antibacterial activities. New quinolones were better inhibitors of DNA gyrase than nalidixic acid and pipemidic acid. We also purified DNA gyrase from a spontaneous nalidixic acid-resistant mutant (M2-5). The gyrases from IID976 and M2-5 were defined as mixtures of subunits As+Bs (s, susceptible) and Ar+Br (r, resistant), respectively. The supercoiling activities of reconstituted Ar+Br and Ar+Bs were more resistant to quinolones than As+Bs and As+Br. These findings indicate that one mechanism of C. freundii resistance against quinolones is resistance modification of the A subunit protein.