scholarly journals Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains

Gene ◽  
2013 ◽  
Vol 514 (2) ◽  
pp. 82-90 ◽  
Author(s):  
Xiaoduo Zhi ◽  
Fenfei Leng
Keyword(s):  
Escherichia Coli ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
Promoter Strength

scholarly journals Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

2021 ◽  
Author(s):  
J Krishna Leela ◽  
Nalini Raghunathan ◽  
J Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Topoisomerase I ◽  
Synthetic Lethality ◽  
Life Forms ◽  
Dna Supercoiling ◽  
Loop Formation ◽  
Chromosomal Dna ◽  
E Coli ◽  
Rnase Hi

Topoisomerase I (Topo I) of Escherichia coli , encoded by topA , acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC -independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including Δ topA , confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued Δ dnaA lethality; (ii) in dnaA + derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by Δ dnaA . We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR. Importance In all life forms, double helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli . That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability, and suggests that in its absence aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

scholarly journals Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

2021 ◽  
Author(s):  
J Gowrishankar ◽  
J Krishna Leela ◽  
Nalini Raghunathan
Keyword(s):  
Escherichia Coli ◽  
Topoisomerase I ◽  
Synthetic Lethality ◽  
Dna Supercoiling ◽  
Loop Formation ◽  
Chromosomal Replication ◽  
E Coli ◽  
Rnase Hi ◽  
Synthetically Lethal ◽  
Chromosome Iii

Topoisomerase I (Topo I) of <Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including ΔtopA>, confer lethality in derivatives of wild-type E. coli strain  MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. colitopA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR.

scholarly journals Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, Topoisomerase I and Fis

2002 ◽  
Vol 35 (6) ◽  
pp. 1413-1420 ◽  
Author(s):  
Dalit Weinstein-Fischer ◽  
Maya Elgrably-weiss ◽  
Shoshy Altuvia
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Topoisomerase I ◽  
Dna Supercoiling

scholarly journals Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

2003 ◽  
Vol 185 (3) ◽  
pp. 1097-1100 ◽  
Author(s):  
Yazmid Reyes-Domínguez ◽  
Gabriel Contreras-Ferrat ◽  
Jesús Ramírez-Santos ◽  
Jorge Membrillo-Hernández ◽  
M. Carmen Gómez-Eichelmann
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Plasmid Dna ◽  
Bimodal Distribution ◽  
Dna Supercoiling ◽  
Wild Type

ABSTRACT Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

Global chromosome topology and the two-component systems in concerted manner regulate transcription in Streptomyces

2021 ◽  
Author(s):  
Martyna Gongerowska-Jac ◽  
Marcin Jan Szafran ◽  
Jakub Mikołajczyk ◽  
Justyna Szymczak ◽  
Magdalena Bartyńska ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Dna Supercoiling ◽  
Complex Life Cycle ◽  
Two Component System ◽  
Bacterial Gene ◽  
Component System ◽  
Global Regulators ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

Bacterial gene expression is controlled at multiple levels, with chromosome supercoiling being one of the most global regulators. Global DNA supercoiling is maintained by the orchestrated action of topoisomerases. In Streptomyces, mycelial soil bacteria with a complex life cycle, topoisomerase I depletion led to elevated chromosome supercoiling, changed expression of significant fraction of genes, delayed growth and blocked sporulation. To identify supercoiling-induced sporulation regulators, we searched for S. coelicolor transposon mutants that were able to restore sporulation despite high chromosome supercoiling. We established that transposon insertion in genes encoding a novel two-component system named SatKR reversed the sporulation blockage resulting from topoisomerase I depletion. Transposition in satKR abolished the transcriptional induction of the genes within the so-called supercoiling-hypersensitive cluster (SHC). Moreover, we found that activated SatR also induced the same set of SHC genes under normal supercoiling conditions. We determined that the expression of genes in this region impacted S. coelicolor growth and sporulation. Interestingly, among the associated products is another two-component system (SitKR), indicating the potential for cascading regulatory effects driven by the SatKR and SitKR two-component systems. Thus, we demonstrated the concerted activity of chromosome supercoiling and a hierarchical two-component signalling system that impacts gene activity governing Streptomyces growth and sporulation.

scholarly journals Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

1996 ◽  
Vol 40 (12) ◽  
pp. 2714-2720 ◽  
Author(s):  
F Blanche ◽  
B Cameron ◽  
F X Bernard ◽  
L Maton ◽  
B Manse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Strong Support ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Dna Relaxation

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

Sign in / Sign up

Export Citation Format

Share Document