DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase

Authors

  • Jacky L. Snoep,

    1. Departments of Molecular Cell Physiology and Mathematical Biochemistry, BioCentrum Amsterdam, Free University, Amsterdam, the Netherlands;
    2. Department of Biochemistry, University of Stellenbosch, South Africa;
    Search for more papers by this author
  • Coen C. van der Weijden,

    1. Departments of Molecular Cell Physiology and Mathematical Biochemistry, BioCentrum Amsterdam, Free University, Amsterdam, the Netherlands;
    Search for more papers by this author
  • Heidi W. Andersen,

    1. Department of Biochemistry, University of Stellenbosch, South Africa;
    2. Section of Molecular Microbiology, Biocentrum, Technical University of Denmark, Lyngby, Denmark;
    Search for more papers by this author
  • Hans V. Westerhoff,

    1. Departments of Molecular Cell Physiology and Mathematical Biochemistry, BioCentrum Amsterdam, Free University, Amsterdam, the Netherlands;
    2. Stellenbosch Institute for Advanced Study, South Africa
    Search for more papers by this author
  • Peter Ruhdal Jensen

    1. Section of Molecular Microbiology, Biocentrum, Technical University of Denmark, Lyngby, Denmark;
    Search for more papers by this author

H. V. Westerhoff, Free University, De Boelelaan 1087, NL-1081 HV Amsterdam, the Netherlands. Fax: + 31 20 4447229, Tel.: + 31 20 4447230, E-mail: hw@bio.vu.nl

Abstract

DNA of prokaryotes is in a nonequilibrium structural state, characterized as ‘active’ DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell34, 105–113]. We here report on a new method for quantifying homeostatic control of the high-energy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomerase I, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomerase I expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem.266, 865–877]. Of the two regulatory mechanisms via which homeostasis is␣conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomerase I (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomerase I or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wild-type Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).

Ancillary