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.
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) Cell 34, 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).
Snoep
Jacky L
JL
Department of Molecular Cell Physiology, BioCentrum Amsterdam, Free University, Amsterdam, the Netherlands.
van der Weijden
Coen C
CC
Andersen
Heidi W
HW
Westerhoff
Hans V
HV
Jensen
Peter Ruhdal
PR
D001483:Base Sequence; D027081:DNA Gyrase / Q000378:metabolism; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D006706:Homeostasis
D017931:DNA Primers; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase