Effects of DNA gyrase inhibitors in Escherichia coli topoisomerase I mutants.

Relaxation of titratable supercoils in bacterial nucleoids was measured following treatment of topA mutants with coumermycin or oxolinic acid, inhibitors of DNA gyrase. Relaxation occurred after treatment of the mutants with either inhibitor. We detected no significant difference in relaxation between topA- and topA+ strains treated with coumermycin. This finding, together with previous observations, supports the idea that relaxation caused by coumermycin probably arises from the relaxing activity of gyrase itself. The source of DNA relaxation caused by oxolinic acid was not identified. Nucleoid supercoiling can be increased by adding oxolinic acid to a strain that carries three topoisomerase mutations: delta topA, gyrB225, and gyrA (Nalr) (S. H. Manes, G. J. Pruss, and K. Drlica, J. Bacteriol. 155:420-423, 1983). We found that this increase in supercoiling requires partial sensitivity to the drug and at the delta topA and gyrA mutations. Full resistance to oxolinic acid in the presence of the delta topA, gyrB225, and gyrA mutations was conferred by an additional mutation that maps at or near gyrB.

DNA supercoiling in gyrase mutants.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.

Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling.

Oxolinic acid reduced RNA synthesis rates whether chromosome supercoiling decreased, increased, or remained unchanged. Thus, inhibition of RNA synthesis by oxolinic acid appears to involve factors other than average DNA supercoiling level. Coumermycin A1 caused RNA synthesis rates to increase or decrease roughly in parallel with DNA supercoiling.

Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes.

Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density. The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322. Thus topoisomerase I is involved in determining the level of supercoiling in bacteria. Three of the topoisomerase I-defective strains were studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.

Differential effects of antibiotics inhibiting gyrase.

Both oxolinic acid and coumermycin A1, inhibitors of DNA gyrase, block DNA synthesis in Escherichia coli. At low concentrations of oxolinic acid, the rate of bacterial DNA synthesis first declines rapidly but then gradually increases. This gradual increase in synthesis rate depended on the presence of wild-type recA and lexA genes; mutations in either gene blocked the increase in synthesis rate. In such mutants, oxolinic acid caused a rapid decline, followed by a slow, further decrease in DNA synthesis rate. Coumermycin A1, however, produced a more gradual decline in synthesis rate which is unaffected by defects in the recA or lexA genes. An additional difference between the two drugs was observed in a dnaA mutant, in which initiation of replication is temperature sensitive. Low concentrations of oxolinic acid, but not coumermycin A1, reduced thermal inhibition of DNA synthesis rate.

DNA gyrase on the bacterial chromosome: possibility of two levels of action.

In previous studies we have shown that oxolinic acid, a specific inhibitor of the A subunit of DNA gyrase, induces DNA cleavage at 100,000-base-pair intervals on the Escherichia coli chromosome. At subsaturating drug concentrations, cleavage is induced at a fraction of these sites and DNA synthesis is partially inhibited. This partial inhibition is surprisingly rapid even when few sites have been inactivated. We now report kinetic measurements suggesting that inactivation of 100,000-base-pair gyrase sites by oxolinic acid does not inhibit DNA synthesis by simply producing barriers to replication fork movement. Slowing the rate of fork movement, thus increasing the time for a fork to reach a barrier, fails to proportionately slow inhibition of DNA synthesis. Moreover, the initial, rapid phase of inhibition is followed by a slower decline that is not accelerated by increasing the frequency of barriers by raising drug concentrations. These data, when added to the observation that additional oxolinic acid-induced cleavage occurs in replicating regions of the chromosome, suggest that gyrase may function at replication forks as well as at 100,000-base-pair intervals on the chromosome.