Inhibition of the gyrA promoter by transcription-coupled DNA supercoiling in Escherichia coli

Samantha Dages,Fenfei Leng,Kelley Dages,Xiaoduo Zhi

doi:10.1038/s41598-018-33089-4

Samantha Dages, Fenfei Leng + Show 2 more

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https://doi.org/10.1038/s41598-018-33089-4

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Journal: Scientific Reports	Publication Date: Oct 3, 2018
Citations: 14	License type: open-access

Affiliation: Florida International University

Abstract

The E. coli gyrA promoter (PgyrA) is a DNA supercoiling sensitive promoter, stimulated by relaxation of DNA templates, and inhibited by (−) DNA supercoiling in bacteria. However, whether PgyrA can be inhibited by transient and localized transcription-coupled DNA supercoiling (TCDS) has not been fully examined. In this paper, using different DNA templates including the E. coli chromosome, we show that transient and localized TCDS strongly inhibits PgyrA in E. coli. This result can be explained by a twin-supercoiled domain model of transcription in which (+) and (−) supercoiled domains are generated around the transcribing RNA polymerase. We also find that fluoroquinolones, such as ciprofloxacin, can substantially increase the expression of the firefly luciferase under the control of the PgyrA coupled to a divergent IPTG-inducible promoter in the presence of IPTG. This stimulation of PgyrA by fluoroquinolones can be also explained by the twin-supercoiled domain model of transcription. This unique property of TCDS may be configured into a high throughput-screening (HTS) assay to identify antimicrobial compounds targeting bacterial DNA gyrase.

Highlights

Using a unique in vivo system, we demonstrated that transient and localized (−) TCDS provided by E. coli RNA polymerase could inhibit the PgyrA at the plasmid and chromosomal levels
We found that fluoroquinolones, such as ciprofloxacin, were able to substantially increase the expression of the firefly luciferase under the control of the PgyrA in the presence of IPTG
100 mL of LB was inoculated with 1 mL of overnight bacterial cell culture at ratio of 1:100 until OD600 = ~0.2. 100 μL of bacterial cell culture was added to 900 μL of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM β-mercaptoethanol)

Summary

Introduction

The expression level of β-galactosidase was measured as described in previous publications[38,49]. 100 mL of LB was inoculated with 1 mL of overnight bacterial cell culture at ratio of 1:100 until OD600 = ~0.2. 100 μL of bacterial cell culture was added to 900 μL of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM β-mercaptoethanol). 60 μL of chloroform and 30 μL of 0.1% SDS were added to lyse the cells. After cell lysates were incubated at 30 °C for 5 minute

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