No abstract

The relative abundance of 88 proteins was measured in extracts from three strains of Escherichia coli K-12 that are isogenic except for the topA and gyrB genes. Mutations in these genes slightly raise or lower, respectively, steady-state DNA supercoiling levels but have little effect on growth rate. Altered protein abundances were observed in the mutant strains relative to wild type. Many proteins exhibited minimum abundance at wild-type supercoiling levels, and other proteins exhibited maximal abundance at relaxed levels. A smaller number showed maximal abundance at elevated levels of supercoiling. These data suggest that small, non-lethal changes in DNA supercoiling can have widespread effects on patterns of gene expression.

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: AtopA, 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 least the AtopA and gyrA mutations. Full resistance to oxolinic acid in the presence of the AtopA, gyrB225, and gyrA mutations was conferred by an additional mutation that maps at or near gyrB.

Treatment of bacterial cells with inhibitors of gyrase at high concentration leads to relaxation of DNA supercoils, presumably through interference with the supercoiling activity of gyrase. Under certain conditions, however, the inhibitors can also increase supercoiling. In the case of coumermycin A1, this increase occurs at low drug concentrations. Oxolinic acid increases supercoiling in a partially resistant mutant. We found that increases in chromosomal DNA supercoiling, which were blocked by treatment with chloramphenicol, were accompanied by an increased expression rate of gyrA. This result is consistent with gyrase being responsible for the increase in supercoiling. In wild-type cells, increases in gyrA expression were transient, suggesting that when supercoiling reaches sufficiently high levels, gyrase expression declines. Oxolinic acid studies carried out with a AtopA strain showed that drug treatment also increased plasmid supercoiling. The levels of supercoiing and topoisomer heterogeneity were much higher when the plasmid contained one of several promoters fused to galK. Since oxolinic acid causes an increase in gyrA expression, it appears that gyrase levels may be important in transcription-mediated changes in supercoiling even when topoisomerase I is absent.

Two cases are described which indicate that RNA polymerase could alter DNA supercoiling. One occurred in a topA mutant in which abnormally high levels of plasmid supercoiling were lowered by rifampin, an inhibitor of the 0 subunit of RNA polymerase. The second case involves suppression of a temperature-sensitive gyrB mutation by a rifampin-resistant allele of rpoB, the gene encoding the i subunit of RNA polymerase. Measurements of chromosomal DNA supercoiling show that the rpoB mutation reduced DNA relaxation.DNA supercoiling in bacteria is controlled by the activities of two enzymes, DNA gyrase and DNA topoisomerase I (for reviews, see references 4 and 25). Gyrase introduces negative supercoils, and topoisomerase I modulates the effects of gyrase by removing them. The level of supercoiling itself appears to be homeostatically regulated by levels of expression of the genes encoding these two enzymes (13, 14, 23; Y. Tse-Dinh and R. Beran, J. Mol. Biol., in press). Even small changes in supercoiling expected to arise from changes in temperature or intercalating dyes appear to be corrected by topoisomerase action (6, 9). Abnormal levels of supercoiling can, however, be generated by mutations in the genes encoding topoisomerases (16,19,20,21,24) and by inhibitors of gyrase (5,11,12,18). We report two examples in which DNA supercoiling was influenced by RNA polymerase. The first involves the ability of rifampin to lower the very high levels of plasmid supercoiling seen in a topA (topoisomerase I) mutant (16), and the second concerns the suppression of a temperature-sensitive gyrB (gyrase) mutation by an rpoB (RNA polymerase) mutation (8).The high levels of negative supercoiling seen in pBR322 when the plasmid is isolated from topA mutants (16) depend on the integrity of the tet gene: insertion or deletion mutations in the tet promoter or in the 5' coding region of tet lower supercoiling (17). To further test the idea that RNA polymerase and transcription are responsible for this phenomenon, we examined the effects of rifampin, an inhibitor of RNA polymerase, on DNA supercoiling. Strain DM800 (3,19,22), transformed with pBR322, was grown to mid-log phase in M9 medium (15) and labeled by growth in tritiated thymidine (10 ,uCi/ml) for about 0.5 cell generations. The cells were then chilled quickly and harvested by centrifugation. For plasmid studies, cells were lysed by incubation with egg white lysozyme followed by dilution into 1.25 volumes of 0.1% Triton X-100-0.05 M EDTA, pH 8. Cellular debris and chromosomal DNA were removed by centrifugation, and the resulting supernatant was deproteinized by incubation with 0.2% sodium dodecyl sulfate-94 jig of proteinase K per ml at 37°C for 30 min. Sedimentation analyses were performed as described previously (5)