Negative supercoiling of bacterial DNA by DNA gyrase influences all metabolic processes involving DNA and is essential for replication. Gyrase supercoils DNA by a mechanism called sign inversion, whereby a positive supercoil is directly inverted to a negative one by passing a DNA segment through a transient double-strand break. Reversal of this scheme relaxes DNA, and this mechanism also accounts for the ability of gyrase to catenate and uncatenate DNA rings. Each round of supercoiling is driven by a conformational change induced by adenosine triphosphate (ATP) binding: ATP hydrolysis permits fresh cycles. The inhibition of gyrase by two classes of antimicrobials reflects its composition from two reversibly associated subunits. The A subunit is particularly associated with the concerted breakage-and-rejoining of DNA and the B subunit mediates energy transduction. Gyrase is a prototype for a growing class of prokaryotic and eukaryotic topoisomerases that interconvert complex forms by way of transient double-strand breaks.