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

Abstract

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.

Figure 1

Experimental design of a pair of divergently coupled transcription units to examine transcription inhibition of P_gyrA by TCDS in vivo. (A) Divergently coupled promoters P_T7A1/O4 and P_gyrA, respectively, control the expression of β-galactosidase (lacZ) and firefly luciferase (luc). (B) The DNA sequence of the pair of divergently coupled promoters, P_T7A1/O4 and P_gyrA. Underlined are P_gyrA and P_T7A1/O4 with −10 and −35 regions. (C,D) Maps of circular plasmid pZXD144 and linear plasmid pZXD150. Winged triangles represent Rho-independent rrnB T1 transcription terminators.

Figure 2

Inhibition of P_gyrA by TCDS for circular plasmid pZXD144 (A,B) and linear plasmid pZXD150 (C, D). The activities of β-galactosidase (Miller’s units) and firefly luciferase (RLU, relative light units) were determined as described under Methods and plotted versus the IPTG concentration. (A,B) E. coli strains MG1655(DE3)ΔlacZ (black squares and lines) and VS111(DE3)ΔlacZ (red circles and lines) carrying pZXD144 were used. (C,D) E. coli strains MG1655(DE3)ΔlacZ (black squares and lines) and VS111(DE3)ΔlacZ (red circles and lines) carrying pZXD150 were used. The standard deviation (SD) was determined according to results from three independent experiments.

Figure 3

Strong inhibition of the supercoiling-sensitive P_gyrA by TCDS on the chromosome. (A,B) TCDS assays for P_gyrA on the chromosome. E. coli strains FL1181 (MG1655(DE3)ΔlacZ attTn7::P_T7A1/O4lacZ-P_gyrAluc; black squares and lines) and FL1182 (VS111(DE3)ΔlacZ attTn7::P_T7A1/O4lacZ-P_gyrAluc; red circles and lines) were used. The activities of β-galactosidase and firefly luciferase were determined as described under Methods and plotted versus the IPTG concentration. (C,D) Effects of novobiocin (C) and ciprofloxacin (D) on P_gyrA of FL1181 (black squares and lines) and FL1182 (red circles and lines) in the absence of IPTG. (E,F) DNA gyrase inhibitors significantly enhanced the expression of firefly luciferase for FL1181 and FL1182 in the presence of IPTG. Overnight cell cultures were diluted 100-fold and grown until OD600 reached ~0.2. Then 0.5 mM of IPTG and various concentrations of ciprofloxacin or other antibiotics were added to the cell cultures. After 30 min incubation, the activities of β-galactosidase and firefly luciferase were determined described under Methods. (C) Ciprofloxacin (CIXP) inhibited the expression of β-galactosidase. (D) CIXP greatly enhanced the expression of firefly luciferase. The standard deviation (SD) was determined according to results from three independent experiments.

Figure 4

The stimulation of expression of firefly luciferase of FL1181 (A) and FL1182 (C) by fluoroquinolones in the presence of 0.5 mM IPTG. CIXP, LVF, EFX, NFX, and novobiocin represent ciprofloxacin, levofloxacin, enrofloxacin, norfloxacin, and novobiocin, respectively. Three bars from left to right represent luciferase activities in the presence of 0, 5, and 10 μM of fluoroquinolones, respectively. (B and D) The inhibition of expression of firefly luciferase by other antibiotics (none gyrase inhibitors) for FL1181 (B) and FL1182 (D). RMP, KM, AMP, and TC represent rifampicin, kanamycin, ampicillin, and tetracycline, respectively. The following are concentrations used in the experiments from left to right: AMP, 0, 150, 300 μM; KM, 0, 40, 80 μM; RMP, 0, 25, 50 μM; TC, 0, 10, 20 μM. The standard deviation (SD) was determined according to results from three independent experiments.

Figure 5

A possible mechanism to explain effects of ciprofloxacin on P_gyrA in the presence of IPTG. In the presence of IPTG (right panel), transcription from P_T7A1/O4 induces significant TCDS and inhibits the expression of firefly luciferase from P_gyrA. However, in the presence of gyrase inhibitor ciprofloxacin, ciprofloxacin stabilizes gyrase-cipro-DNA complex that blocks transcription from P_T7A1/O4. The (−) supercoils behind RNA polymerase are not formed. As a result, the expression of firefly luciferase is “enhanced.”