We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnoA-dnaN-recF-gyrB-gyrA, is similar to that found in the BaciUus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640-and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.DNA gyrase, bacterial topoisomerase II, catalyzes ATPdependent DNA supercoiling and is essential for bacterial growth, being involved in DNA replication, transcription, and recombination (9, 37). The Escherichia coli enzyme consists of two A and two B subunits, the 97-and 90-kDa products of the gyrA (48 min) and gyrB genes (82 min), respectively (37). The subunits provide different functions in the DNA strand passage process by which gyrase mediates topological changes in DNA (20). The GyrA subunits form a transient double-strand break in DNA by linking to each complementary DNA strand via . A second DNA duplex is then passed through the gyrase-DNA gate in a process coupled to ATP binding and hydrolysis by the B subunits (20). Several lines of evidence show that the E. coli gyrase subunits are each organized into domains with discrete enzymatic functions (26), as first suggested by studies of a fragment of the gyrase B protein (7).Two classes of drug act by targeting DNA gyrase. Antibacterial 4-quinolones, such as ciprofloxacin, interrupt DNA breakage and resealing by the A subunits (8), whereas the coumarin drugs novobiocin and coumermycin interfere with ATP utilization by the B proteins (10, 32). Gyrase inhibitors have been widely used, particularly against Staphylococcus aureus, a gram-positive organism for which few clinically effective agents are available (13). Both ciprofloxacin and novobiocin are active against S. aureus, including methicillin-resistant strains (11,13,25,36), although resistance to fluoroquinolones has rapidly emerged (16,28,29 (2), and Neisseria gonorrhoeae (31), and from Pseudomonas putida (24).
MATERIALS AND METHODSBacterial strains, plasmids, reagents. Cloning of the gyrBgyrA locus from S. aureus 81231 as overlapping inserts contained in plasmids pRH1, pRH2, and pRH3 has been described previously (14). Propagation of the plasmids was in E. coli DH5ao [F-endA1 hsdR17 (rK-MK+) supE44 thi-J recAI gyrA96 rell ]. E. coli XL-1 Blue {endAl hsdRJ7 (rK-MK+) supE44 thi-J X-recAl gyrA96 reLAl (lac) [F' proAB lacIVAMI5 TnlO(Tetr)