Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Plasmid DNA supercoiling and gyrase activity in Escherichia coli wild-type and rpoS stationary-phase cells

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.     

Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity

DNA gyrase from Bacillus subtilis 168 was purified by affinity chromatography on novobiocin-Sepharose and shown to consist of two subunits, A and B, with molecular weights of 100,000 and 85,000, respectively. The B subunits, which contains novobiocin-sensitive. ATPase activity, could complement the gyrA protein of Escherichia coli. No complementation was detected between the A subunit and the E. coli gyrB protein.     

DNA gyrase on the bacterial chromosome. Oxolinic acid-induced DNA cleavage in the dnaA-gyrB region

Oxolinic acid forms complexes with gyrase and DNA in such a way that subsequent denaturation of gyrase reveals DNA cleavage. Cleavage sites were mapped in a 10,000 base-pair region of the Escherichia coli chromosome containing the dnaA, dnaN, recF, and gyrB genes. Twenty-four cleavage sites were identified. The sites were cleaved at different frequencies, with the most frequent cleavage occurring within gyrB. Not all sites were equally sensitive to oxolinic acid concentration, some sites exhibited an altered cleavage frequency when the gyrB225 delta topA mutant strain DM800 was compared with wild-type cells, and coumermycin selectively changed the cleavage frequency at a few sites in the mutant strain DM800. These perturbations appear to alter the frequency of cleavage at a site but not the location of the site. The availability of many sites of differing strengths may be an important factor in the ability of gyrase to fine-tune the level of supercoiling or provide local swivels in bacterial DNA.