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

The E. coli gyrA promoter (P_gyrA) is a DNA supercoiling sensitive promoter, stimulated by relaxation of DNA templates, and inhibited by (-) DNA supercoiling in bacteria. However, whether P_gyrA 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 P_gyrA 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 P_gyrA coupled to a divergent IPTG-inducible promoter in the presence of IPTG. This stimulation of P_gyrA 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.

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