Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli

DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV acting in concert with topoisomerase I and gyrase , makes an important contribution to the steady-state level of supercoiling in Escherichia coli, Following inhibition of gyrase, topoisomerase IV alon e relaxed plasmid DMA to a final supercoiling density (a) of -0.015 at an i nitial rate of 0.8 links min-l. Topoisomerase I relaxed DNA at a faster rat e, 5 links min-l, but only to a a of -0.05, Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level a s in a mutant lacking topoisomerase I activity (to sigma = -0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase 1 and topoisomerase IV caused the DNA to become hyper-negati vely supercoiled (sigma = -0.09), greatly stimulating transcription from th e supercoiling sensitive leu-500 promoter and increasing the number of supe rcoils trapped by A integrase site-specific recombination.