Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5-to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5-to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.A major human infectious disease, tuberculosis (TB) is estimated to affect approximately one-third of the world's population, and 95% of cases occur in developing countries (15,30,31). Current estimates show that approximately 9.4 million new cases and nearly 1.7 million deaths from TB occur each year, and TB remains a major cause of premature death (36).The increased incidence of multidrug-resistant (MDR) TB (TB resistant to more than two anti-TB drugs, including rifampin and isoniazid [35]) has hampered the treatment and control of TB and is associated with an increase in mortality rates in people with TB (3,37,40). Consequently, the required drug dosage for the treatment of TB has dramatically increased (38), and fluoroquinolones (FQs) are now considered to be important second-line anti-TB agents (13,20