scholarly journals Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling.

1986 ◽  
Vol 83 (23) ◽  
pp. 8952-8956 ◽  
Author(s):  
G. J. Pruss ◽  
K. Drlica
Keyword(s):  
Plasmid Dna ◽  
Topoisomerase I ◽  
Dna Supercoiling

scholarly journals Uncoupling of the Diurnal Growth Program by Artificial Genome Relaxation in Synechocystis sp. PCC 6803

2021 ◽  
Author(s):  
Anna Behle ◽  
Maximilian Dietsch ◽  
Louis Goldschmidt ◽  
Wandana Murugathas ◽  
David Brandt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Plasmid Dna ◽  
Topoisomerase I ◽  
Metabolic Flux ◽  
Dna Supercoiling ◽  
Dark Cycle ◽  
Dark Light ◽  
Wildtype Cell ◽  
Transcription And Replication ◽  
Pcc 6803

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knock-down of gyrase subunits and overexpression of topoisomerase I, and characterized the phenotypes. Cell division was blocked, most likely due to inhibition of genomic but not plasmid DNA replication. Cell growth continued to 4-5x of the wildtype cell volume, and metabolic flux was redirected towards glycogen in the topoI overexpession strain. Topoisomerase I induction initially lead to down-regulation of GC-rich and up-regulation of AT-rich genes. The response quickly bifurcated and four diurnal co-expression cohorts (dawn, noon, dusk and night) all responded differently, in part with a circadian (≈24 h) pattern. We suggest a model where energy- and gyrase-gated transcription of growth genes at the dark/light transition (dawn) generates DNA supercoiling which then directly facilitates DNA replication and initiates the diurnal transcriptome program.

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 The Global Regulatory Cyclic AMP Receptor Protein (CRP) Controls Multifactorial Fluoroquinolone Susceptibility in Salmonella enterica Serovar Typhimurium

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stefani C. Kary ◽  
Joshua R. K. Yoneda ◽  
Stephen C. Olshefsky ◽  
Laura A. Stewart ◽  
Steven B. West ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Salmonella Enterica ◽  
Topoisomerase I ◽  
Multiple Drug Resistance ◽  
Regulatory Protein ◽  
Dna Supercoiling ◽  
Receptor Protein ◽  
Multiple Drug ◽  
Topoisomerase Iv ◽  
Fluoroquinolone Antibiotics

ABSTRACT Fluoroquinolone antibiotics are prescribed for the treatment of Salmonella enterica infections, but resistance to this family of antibiotics is growing. Here we report that loss of the global regulatory protein cyclic AMP (cAMP) receptor protein (CRP) or its allosteric effector, cAMP, reduces susceptibility to fluoroquinolones. A Δcrp mutation was synergistic with the primary fluoroquinolone resistance allele gyrA83, thus able to contribute to clinically relevant resistance. Decreased susceptibility to fluoroquinolones could be partly explained by decreased expression of the outer membrane porin genes ompA and ompF with a concomitant increase in the expression of the ciprofloxacin resistance efflux pump gene acrB in Δcrp cells. Expression of gyrAB, which encode the DNA supercoiling enzyme GyrAB, which is blocked by fluoroquinolones, and expression of topA, which encodes the dominant supercoiling-relaxing enzyme topoisomerase I, were unchanged in Δcrp cells. Yet Δcrp cells maintained a more relaxed state of DNA supercoiling, correlating with an observed increase in topoisomerase IV (parCE) expression. Surprisingly, the Δcrp mutation had the unanticipated effect of enhancing fitness in the presence of fluoroquinolone antibiotics, which can be explained by the observation that exposure of Δcrp cells to ciprofloxacin had the counterintuitive effect of restoring wild-type levels of DNA supercoiling. Consistent with this, Δcrp cells did not become elongated or induce the SOS response when challenged with ciprofloxacin. These findings implicate the combined action of multiple drug resistance mechanisms in Δcrp cells: reduced permeability and elevated efflux of fluoroquinolones coupled with a relaxed DNA supercoiling state that buffers cells against GyrAB inhibition by fluoroquinolones.

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

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