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 alone relaxed plasmid DNA to a final supercoiling density () of ؊0.015 at an initial rate of 0.8 links min ؊1 . Topoisomerase I relaxed DNA at a faster rate, 5 links min ؊1 , but only to a of ؊0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to ‫؍‬ ؊0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled ( ‫؍‬ ؊0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by integrase site-specific recombination.

No abstract

Palindromic Units (PU or REP) were defined as DNA sequences of 40 nucleotides highly repeated on the genome of Escherichia coli and other Enterobacteriaceae. PU are found in clusters of up to six occurrences always localized in extragenic regions. By sorting the DNA sequences of the known PU containing regions into different classes, we show here for the first time that, besides the PU themselves, each PU clusters contains a number of other conserved sequence motifs. Seven such motifs were identified with the present list of PU regions. Remarkably, each PU cluster is exclusively composed of a mosaic combination of PU and of these other sequence motifs. We demonstrate directly by hybridization experiments that one of these motifs (called L) is indeed present at a large number of copies on the Escherichia coli chromosome and that its distribution follows the same species specificity as PU sequences themselves. We propose that the mosaic pattern of motif combination in PU clusters reveals a new type of bacterial genetic element which we propose to call BIME for Bacterial Interspersed Mosaic Element. The Escherichia coli genome contains about 500 BIME.