DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus

Abstract

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli

“…However, under specific growth conditions, others have found that acrA lesions are compensatory for deletion of topA. Dorman et al (6) showed that AtopA could be transduced directly into an acrA mutant background when cells are grown in a low-osmolarity medium. Nothing is known about why acrA mutations follow deletion of topA or how these acrA mutants permit loss of topA during growth in a low-osmolarity medium.…”

Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition

“…The type II topoisomerases are thought to be essential for bacterial growth because of inducible lethality in temperature-sensitive mutants (16,17). The type I topoisomerases are not essential for bacterial growth, because their loss can be compensated for by alterations in type II topoisomerase genes (4,5).…”

“…Although all of these topoisomerases are capable of changing the topology of DNA by a cleaving and rejoining step, each enzyme appears to possess a favored reaction in vitro. Type I topoisomerases, topoisomerase I and topoisomerase III, show efficient relaxing and decatenating activity, respectively, in the absence of ATP (2,3,5,11,29,30,34). Of the type II topoisomerases, DNA gyrase is unique in its ability to supercoil relaxed DNA in the presence of ATP (9) and topoisomerase IV decatenates catenated DNA in vitro (28).…”

“…Although all of these topoisomerases are capable of changing the topology of DNA by a cleaving and rejoining step, each enzyme appears to possess a favored reaction in vitro. Type I topoisomerases, topoisomerase I and topoisomerase III, show efficient relaxing and decatenating activity, respectively, in the absence of ATP (2,3,5,11,29,30,34 subunits of DNA gyrase), respectively (16). Similar sequences are located especially around the region of DNA gyrase known as the "quinolone resistance-determining region."…”

Roles of Topoisomerases in Maintaining Steady-state DNA Supercoiling in Escherichia coli

“…(ii) DNA isolated from topoisomerase I mutant strains generally is more (Ϫ) supercoiled than that from wild-type strains (43)(44)(45). (iii) For topoisomerase I deletion mutants to be viable, there must be compensatory mutations elsewhere, many of which mapped to the gyrase genes (23,24,46,47). (iv) Inhibition of DNA gyrase in cells leads to DNA relaxation and, in those DNA molecules being transcribed, (ϩ) supercoil accumulation.…”