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Chromosomal supercoiling in Escherichia coli
Author(s) -
Miller† William G.,
Simons Robert W.
Publication year - 1993
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1993.tb00939.x
Subject(s) - dna supercoil , biology , lac operon , dna gyrase , escherichia coli , genetics , operon , nucleoid , transposable element , microbiology and biotechnology , fusion gene , gene , dna replication , mutant
Summary The Escherichia coli chromosome is compacted into 40‐50 negatively supercoiled domains. It has been proposed that these domains differ in superhelical density. Here, we present evidence that this is probably not the case. A modified Tn 10 transposable element was inserted at a number of locations around the E. coli chromosome. This element, mTn 10‐plac‐lacZ + , contains the lac operon promoter, plac , whose activity increases with increasing superhelical density, fused to a lacZ + reporter gene. Although mTn 10‐plac‐lacZ + fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA , which has a reciprocal response to changes in superhelical density. Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn 10‐plac‐lacZ + and mTn 10‐pgyrA‐lacZ + fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density. Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling. The approximately threefold difference in mTn 10‐plac‐lacZ ‐ and mTn 10‐pgyrA‐lacZ + fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication. Together, these results strongly suggest that the E. coli chromosomal domains do not differ in functional superhelical density.