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Chromosomal supercoiling in Escherichia coli

W G Miller1, R W Simons

  • 1Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.

Molecular Microbiology
|November 1, 1993
PubMed
Summary

Escherichia coli chromosomal domains likely share similar superhelical density. Reporter gene expression differences are not due to varying supercoiling levels, suggesting uniform DNA superhelix density across the E. coli chromosome.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • The bacterial chromosome, like that of Escherichia coli, is organized into supercoiled domains.
  • It has been hypothesized that these domains possess distinct superhelical densities.

Purpose of the Study:

  • To investigate whether different domains of the Escherichia coli chromosome exhibit varying superhelical densities.
  • To determine if functional superhelical density is uniform across the E. coli chromosome.

Main Methods:

  • Insertion of a modified Tn10 transposable element (mTn10-plac-lacZ+) containing a supercoiling-sensitive promoter (plac) and a reporter gene (lacZ+) at multiple chromosomal locations.
  • Comparison of reporter gene expression using both plac and a supercoiling-insensitive promoter (pgyrA).
  • Assessment of reporter gene expression changes in response to topoisomerase mutations and coumermycin treatment, as well as under anaerobic growth and osmotic shock conditions.

Main Results:

  • Fusion expression varied up to threefold at different insertion sites, but relative expression levels remained consistent when switching between plac and pgyrA promoters.
  • Topoisomerase mutations and coumermycin altered reporter gene expression as expected, confirming the system's responsiveness to supercoiling changes.
  • Expression levels were unaffected by anaerobic growth or osmotic shock, and observed differences were attributed to regional copy number variations from bidirectional replication.

Conclusions:

  • The functional superhelical density is likely uniform across different domains of the Escherichia coli chromosome.
  • Observed variations in reporter gene expression are not attributable to differences in DNA supercoiling levels between chromosomal domains.

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