FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>

ABSTRACT DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here, we report a method for high-throughput mea...

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Autores principales: Alexandre Duprey, Eduardo A. Groisman
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Publicado: American Society for Microbiology 2020
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Acceso en línea:https://doaj.org/article/65db34ab76d84080becb993baa6e4e72
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spelling oai:doaj.org-article:65db34ab76d84080becb993baa6e4e722021-11-15T15:56:44ZFEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>10.1128/mBio.01053-202150-7511https://doaj.org/article/65db34ab76d84080becb993baa6e4e722020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01053-20https://doaj.org/toc/2150-7511ABSTRACT DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here, we report a method for high-throughput measurement of bacterial DNA supercoiling in vivo. Fluorescent evaluation of DNA supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling and the gene for a red fluorescent protein transcribed by a constitutive promoter as the internal standard. Using FEDS, we uncovered single-cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low-mean/high-variance DNA supercoiling subpopulation rather than from a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases. IMPORTANCE DNA represents the chemical support of genetic information in all forms of life. In addition to its linear sequence of nucleotides, it bears critical information in its structure. This information, called DNA supercoiling, is central to all fundamental DNA processes, such as transcription and replication, and defines cellular physiology. Unlike reading of a nucleotide sequence, DNA supercoiling determinations have been laborious. We have now developed a method for rapid measurement of DNA supercoiling and established its utility by identifying a novel regulator of DNA supercoiling in the bacterium Salmonella enterica as well as behaviors that could not have been discovered with current methods.Alexandre DupreyEduardo A. GroismanAmerican Society for MicrobiologyarticleDNA gyraseDNA topologyfeedback loopgene transcriptionMicrobiologyQR1-502ENmBio, Vol 11, Iss 4 (2020)
institution DOAJ
collection DOAJ
language EN
topic DNA gyrase
DNA topology
feedback loop
gene transcription
Microbiology
QR1-502
spellingShingle DNA gyrase
DNA topology
feedback loop
gene transcription
Microbiology
QR1-502
Alexandre Duprey
Eduardo A. Groisman
FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>
description ABSTRACT DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here, we report a method for high-throughput measurement of bacterial DNA supercoiling in vivo. Fluorescent evaluation of DNA supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling and the gene for a red fluorescent protein transcribed by a constitutive promoter as the internal standard. Using FEDS, we uncovered single-cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low-mean/high-variance DNA supercoiling subpopulation rather than from a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases. IMPORTANCE DNA represents the chemical support of genetic information in all forms of life. In addition to its linear sequence of nucleotides, it bears critical information in its structure. This information, called DNA supercoiling, is central to all fundamental DNA processes, such as transcription and replication, and defines cellular physiology. Unlike reading of a nucleotide sequence, DNA supercoiling determinations have been laborious. We have now developed a method for rapid measurement of DNA supercoiling and established its utility by identifying a novel regulator of DNA supercoiling in the bacterium Salmonella enterica as well as behaviors that could not have been discovered with current methods.
format article
author Alexandre Duprey
Eduardo A. Groisman
author_facet Alexandre Duprey
Eduardo A. Groisman
author_sort Alexandre Duprey
title FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>
title_short FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>
title_full FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>
title_fullStr FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>
title_full_unstemmed FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <italic toggle="yes">In Vivo</italic>
title_sort feds: a novel fluorescence-based high-throughput method for measuring dna supercoiling <italic toggle="yes">in vivo</italic>
publisher American Society for Microbiology
publishDate 2020
url https://doaj.org/article/65db34ab76d84080becb993baa6e4e72
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