Rifampin and rpoB mutations can alter DNA supercoiling in Escherichia coli

Abstract

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)

Gyrase inhibitors can increase gyrA expression and DNA supercoiling

“…Promoter-dependent increases in negative supercoiling had been reported previously for the tet gene in pBR322 in strain DM800 (32), from which GP200 was derived. In the case of DM800, rifampin treatment eliminates the high level of supercoiling (6), and it is likely that with both DM800 and GP200 the increase in supercoiling is stimulated by transcription. Our experiments with strain GP200 extend the previous studies in two ways.…”

The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli

“…However, DNA synthesis in both systems is sensitive to rifampicin, suggesting a second unknown capacity of rifampicin (38). It is known that incubation of cells with rifampicin results in a decrease in the sedimentation rate of nucleoids, suggesting a decrease in the overall extent of supercoiling of the nucleoids (39,40). We suggest the possibility that rifampicin, which shuts down all transcriptions on the chromosome, brings about a drastic global change in the nucleoid structure, severely altering the topological structure of oriC.…”

A Biochemical Mechanism for Nonrandom Mutations and Evolution

“…Some environmental perturbations affect plasmid systems and chromosomes in a similar manner, while some apparently do not (25). Transcription both responds to and promotes changes in supercoiling.…”