Viability of Escherichia coli topA Mutants Lacking DNA Topoisomerase I

Abstract

The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42°C without any compensatory mutations. These topA ؊ cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. The type IA subfamily of DNA topoisomerases, some of the extensively studied examples are E. coli DNA topoisomerases I and III, yeast DNA topoisomerase III, Drosophila, and mammalian DNA topoisomerases III␣ and III␤, are found in all living organisms (1-3). This universal presence suggests a key cellular role of these enzymes that cannot be fulfilled by either the type IB or type II subfamily of DNA topoisomerases.

Analysis of Pleiotropic Transcriptional Profiles: a Case Study of DNA Gyrase Inhibition

“…The E. coli strain carrying the deletion of the topA allele without the compensatory mutations has been reported recently [21]. We confirmed that transduction of the DtopA into the MG1655 background was not accompanied by the compensatory mutations in the gyrA or gyrB genes or by any changes in the tolC-par and topB regions of the chromosome (unpublished data).…”

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

“…These results are consistent with our previously published results for T7 RNA polymerase where the strong T7 RNA polymerase efficiently drove plasmid DNA templates to hypernegatively supercoiled status even when the transcriptional machinery did not couple to translation and membrane insertion . We noticed that these results appear inconsistent with the previously published results showing that plasmid hypernegative supercoiling by E. coli RNA polymerase required the anchoring or insertion of the coupled transcription-translation complex into the cytoplasmic membrane Lynch and Wang, 1993;Ma et al, 1994;Pruss and Drlica, 1986;Stupina and Wang, 2005). However, a careful analysis showed that both situations can be explained by the "twin-supercoiled-domain" model of transcription where the friction force applied to E. coli RNA polymerase is different for promoters with different strengths.…”

“…In this case, in order to generate "twin-supercoileddomains," the transcriptional ensemble has to anchor to the bacterial cytoplasmic membrane through co-transcriptional synthesis of polypeptide encoding membrane proteins to maximize friction resistance. This interpretation explains why TCDS for plasmid pBR322 and its derivatives carrying the P tet promoter depends on the expression of a membrane-insertion tetracycline resistance protein in E. coli topA strains Lynch and Wang, 1993;Ma et al, 1994;Pruss and Drlica, 1986;Stupina and Wang, 2005). For strong promoters, such as P T7A1/O4 and P tac , E.…”

“…As mentioned above, we simultaneously introduced the linear plasmid pZXD51 and a supercoiling-reporter plasmid into E. coli topA strains VS111 and DM800. Since previous studies showed that hypernegative supercoiling of plasmid pBR322 and its derivatives is dependent on the expression of the co-transcription and translation of membrane-associated tet gene Lynch and Wang, 1993;Ma et al, 1994;Pruss and Drlica, 1986;Stupina and Wang, 2005), we decided to use a set of five supercoiling-reporter plasmids that carry a tet gene under the control of an IPTGinducible promoter with different strengths, i.e., promoters P T7A1/O4 , P tac , P lacUV5 , P lac , and P lacL8 . As expected, our RT-PCR experiments (Figure 2.2) demonstrated that the transcription level of E. coli strains harboring different plasmids after IPTG induction is correlated with promoter strength in vivo .…”

“…Inside a bacterium, DNA gyrase and topoisomerase I act differentially on positively and negatively supercoiled domains (Wang, 1971;Gellert et al, 1976), therefore the supercoiling state of intracellular DNA is expected to be regulated by several processes, for instance, the transcription process which produces negative and positive supercoils at equal rates, the diffusion pathways which allow the cancellation of negative and positive supercoils, and the actions of DNA topoisomerases such as DNA topoisomerase I-catalyzed relaxation for negatively supercoiled DNA as well as gyrasecatalyzed negative supercoiling There are several studies conducted to support the "twin-supercoiled-domain" model of transcription Tsao et al, 1989;Dröge and Nordheim, 1991;Dayn et al, 1992;Rahmouni and Wells, 1992;Dunaway and Ostrander, 1993;Lynch and Wang, 1993;Ma et al, 1994;Stupina and Wang, 2005;. Liu and Wang's model also suggested that in a dilute aqueous solution, the friction force applying to the transcription complex was too small to generate significant supercoiling of the DNA template that only contain one transcription unit .…”