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

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.

can strongly activate P leu-500 . The activation of P leu-500 is dependent of the promoter strength and the length of RNA transcripts, unique properties of TCDS as predicted by the twin-supercoiled domain mechanism. We also demonstrated that TCDS could be generated on topologically open DNA molecules in E. coli cells. These results suggest that topological boundaries or barriers are not necessary for the generation of TCDS in vivo.
The E. coli gyrA promoter (P gyrA ) is another supercoiling sensitive promoter and stimulated by relaxation of DNA templates 7,39,40 . Early mutation studies showed that the stimulation of P gyrA stems from a 20 bp DNA sequence around the −10 region of P gyrA 39,40 . Since this 20 bp DNA sequence is intrinsically bent or curved 41 , it is possible that the DNA bend or curvature functions as a supercoiling sensor for the activation by DNA relaxation 41 . Nevertheless, whether P gyrA can be inhibited by TCDS has not been examined. Here, using different DNA templates including the E. coli chromosome, we show that transient and localized (−) TCDS is able to strongly inhibit P gyrA in E. coli. We also found that fluoroquinolones, such as ciprofloxacin, were able to substantially increase the expression of the firefly luciferase controlled by P gyrA coupled to a divergent IPTG-inducible promoter in the presence of IPTG. This unique property of TCDS may be used to screen and identify antimicrobial compounds targeting bacterial DNA gyrase.

Results and Discussion
In our previous studies 38 , using an in vivo system that contains E. coli topA strain VS111(DE3)ΔlacZ or wild-type strain MG1655(DE3)ΔlacZ and a circular or linear plasmid DNA template, we demonstrated that transient and localized TCDS from a divergently-coupled transcription unit potently activated the supercoiling-sensitive promoter P leu-500 . In this study, we decided to utilize this system to examine whether and how TCDS inhibits a different supercoiling-sensitive promoter P gyrA . For this purpose, we substituted P leu-500 with P gyrA divergently coupled to the strong IPTG-inducible promoter P T7A1/O4 (Fig. 1). The distance between these two promoters is 92 bp (Fig. 1A). As shown in Fig. 1C,D, we used 2 sets of 4 Rho-independent, rrnB T1 transcription terminators to block transcription from P T7A1/O4 and P gyrA , respectively. In this case, transcription is restricted to a selected region of the plasmids 22 . Circular plasmid pZXD144 and linear plasmid pZXD150 were used to transform VS111(DE3)ΔlacZ or MG1655(DE3)ΔlacZ. After IPTG was added to E. coli cells in the early log phase, luciferase activities were used to monitor the inhibition of P gyrA . Results in Fig. 2 show that TCDS strongly inhibits the supercoiling-sensitive P gyrA for both circular and linear plasmids. For example, TCDS from E. coli RNA polymerase on pZXD144 inhibited 53% and 68% of P gyrA in VS111(DE3)ΔlacZ and MG1655(DE3)ΔlacZ, respectively, comparing with the activities of P gyrA in the absence of IPTG (Fig. 2B). TCDS on pZXD150 inhibited 42% and 63% of P gyrA in VS111(DE3) ΔlacZ and MG1655(DE3)ΔlacZ, respectively (Fig. 2D). Due to the fact that linear DNA templates cannot be permanently supercoiled 42 , these results unambiguously demonstrated that transient and localized TCDS, rather than global supercoiling, inhibits the divergently coupled P gyrA . Interestingly, for circular plasmid pZXD144, the expression level of β-galactosidase is always higher in MG1655(DE3)ΔlacZ than that in VS111(DE3)ΔlacZ in the absence or presence of IPTG ( Fig. 2A), which is consistent with our previously published results 43 . In contrast, for linear plasmid pZXD150, the expression level of β-galactosidase is lower in MG1655(DE3)ΔlacZ comparing with that in VS111(DE3)ΔlacZ (Fig. 2C). These results suggest that DNA supercoiling plays some roles in regulating the activities of P T7A1/O4 38 . Please note that each E. coli cell carries approximate 1 copy of a linear plasmid, the overall expression levels of firefly luciferase are much lower for linear plasmids 38 . Since the topA strain VS111 is a DNA topoisomerase I deletion strain, it should have greater supercoiling fluctuations when disturbed by TCDS. As a result, P gyrA should be more sensitive to TCDS. Indeed, our results showed that P gyrA is more sensitive to the IPTG concentration, indicating that it is more sensitive to TCDS ( Fig. 2B and D). Next, we examined how TCDS inhibits P gyrA on the E. coli chromosome. First, we placed a ~5 kb DNA fragment carrying the divergently coupled P gyrA and P T7A1/O4 promoters (Fig. 1A) into the attTn7 site of the E. coli chromosome ( Fig. S1; the 84.2 min of the E. coli chromosome 44 ) using a procedure of transposon Tn7 45 to yield a wild type strain FL1181 (MG1655(DE3)ΔlacZ attTn7::P T7A1/O4 lacZ-P gyrA luc) and a topA strain FL1182 (VS111(DE3)ΔlacZ attTn7::P T7A1/O4 lacZ-P gyrA luc). Due to technical difficulties, the four T1 transcription terminators were not included in these constructs. Similar to results for plasmid DNA templates as shown above, transcription by E. coli RNA polymerase can substantially inhibit transcription from P gyrA on the E. coli chromosome (Fig. 3A,B). For example, TCDS was able to inhibit 24% and 47% of P gyrA in FL1181 and FL1182, respectively. Interestingly, in the absence of IPTG, P gyrA in FL1182 is more active than that in FL1181 (Fig. 3B). As demonstrated previously 43 , in the absence of IPTG, P T7A1/O4 is much more active in the wildtype strain MG1655 that that in the topA strain VS111. Although the DNA templates may be more negatively supercoiled globally in VS111, the localized supercoiling around P gyrA in the wildtype strain MG1655 should be more negatively supercoiled than that in VS111 due to TCDS. In this way, the expression level of luciferase in VS111 should be higher than that in MG1655 in the absence of IPTG.
Since it was shown that gyrase inhibitors, such as coumermycin, quinolones, and novobiocin, are able to induce the expression of gyrA and gyrB in bacteria 46,47 , we also treated FL1181 and FL1182 with two gyrase inhibitors, novobiocin and ciprofloxacin, and examined whether these two gyrase inhibitors are able to increase the firefly luciferase expression under the control of P gyrA . At the early exponential stage, novobiocin slightly enhanced the expression of firefly luciferase in FL1181 (Fig. 3C) and did not have much effect on the expression of firefly luciferase in the topA strain FL1182 (Fig. 3C). Ciprofloxacin at low concentrations slightly stimulated the expression of firefly luciferase for both strains (Fig. 3D; the differences appear to be statistically insignificant) and inhibited the expression of firefly luciferase in FL1181 at 50 μM (Fig. 3D). Intriguingly, in the presence of IPTG, the stimulation of firefly luciferase expression by ciprofloxacin was significantly amplified (Fig. 3F) although ciprofloxacin at high concentrations completely inhibited the expression of β-galactosidase for both strains (Fig. 3E). We noticed some differences between these two E. coli strains. For the wild type strain FL1181, the stimulation of firefly luciferase expression by ciprofloxacin decreased at higher concentrations, i.e., 20 and 50 μM. For topA strain FL1182, however, the stimulation by ciprofloxacin plateaued at 10 μM and stayed high at 50 μM. These results suggest that topoisomerase I plays a role in the regulation of P gyrA activities in E. coli. We further tested several other gyrase inhibitors including levofloxacin, norfloxacin, enrofloxacin, and novobiocin, and found that only fluoroquinolones dramatically stimulated the expression of firefly luciferase in FL1181 and FL1182 in the presence of IPTG ( Fig. 4A and C). Novobiocin's effect on the expression of firefly luciferase is negligible for both strains ( Fig. 4A and C). At the tested concentrations, i.e., 5 and 10 µM, these fluoroquinolones slightly inhibit the growth of the two E. coli strains (Fig. S2). We also tested several other types of antibiotics, such as transcription inhibitors (rifampicin), protein synthesis inhibitors (kanamycin and tetracycline), and cell wall synthesis inhibitors (ampicillin), and found that all these antibiotics inhibited the expression of firefly luciferase in FL1181 and FL1182 (Figs 4B,D and S3). These results suggest that the enhancement of the expression of firefly luciferase is specific for gyrase inhibitors, especially for fluoroquinolones. These results also suggest that this stimulation assay can be used to identify antibiotics targeting bacterial DNA gyrase.
We believe that the twin supercoiled domain model of transcription 24 can explain why gyrase inhibitors are able to stimulate the expression of firefly luciferase in FL1181 and FL1182. At the early exponential phase, RNA polymerase is actively transcribing genes along the E. coli chromosome, introducing localized DNA supercoiling around these genes, and remodeling the chromosome. For FL1181 and FL1182, the divergently coupled P gyrA and P T7A1/O4 promoters with the luc and lacZ genes are located at 84.2 min of the E. coli chromosome near the seven rRNA operons 48 . Since the E. coli RNA polymerase transcribes along these seven rRNA operons away from 84.2 min of the E. coli chromosome, transcription should introduce significant amounts of (−) supercoils to this region. As a result, P gyrA is repressed. For the wild type strain FL1181, in the presence of novobiocin, DNA gyrase is no longer capable of removing (+) supercoiled domain generated during transcription. Topoisomerase I, on the other hand, relaxes (−) supercoiled domain. In this way, DNA templates including the chromosome should be more relaxed, which resulted in the stimulation of the expression of firefly luciferase under the control of P gyrA (Fig. 3C). Since the topA strain FL1182 does not have DNA topoisomerase I to remove (−) supercoiled domain, the DNA supercoiling status in FL1182 will not fluctuate significantly in the presence of novobiocin. This is the reason why novobiocin did not greatly affect the expression of firefly luciferase in FL1182 (Fig. 3C). Ciprofloxacin is a different DNA gyrase inhibitor and forms gyrase-cipro-DNA complexes that cause the termination of transcription for both FL1181 and FL1182 (Fig. 3E). The (−) supercoiled domain should not be formed. As a result, ciprofloxacin was able to "stimulate" the expression of firefly luciferase for both strains (Fig. 3D).
Regarding why fluoroquinolones, in the presence of IPTG, are able to enhance the expression of firefly luciferase ( Fig. 4A and C), we favor the model depicted in Fig. 5 for explanation. In the presence of IPTG, transcription initiated from the strong P T7A1/O4 produces a significant amount of (−) supercoils behind the RNA polymerase and as a result inhibits the expression of firefly luciferase by P gyrA (Fig. 3B). However, ciprofloxacin stabilizes gyrase-cipro-DNA complexes for those DNA gyrases that remove the (+) supercoiled domain in front of RNA polymerase. As a result, transcription from P T7A1/O4 is terminated (Fig. 3E) and the (−) supercoiling domain behind the RNA polymerase is not formed. Because P gyrA is a weak promoter and transcription from P gyrA should not produce significant amounts of (+) supercoils in front of RNA polymerase, gyrase-cipro-DNA complexes are not formed. In this scenario, ciprofloxacin will not be able to inhibit the expression of firefly luciferase. In contrast, the (−) DNA supercoiled domain from the divergently coupled P T7A1/O4 is not formed, the expression of firefly luciferase is greatly "enhanced" (Fig. 3F). Because novobiocin only inhibits DNA gyrase activities and does not form gyrase-novobiocin-DNA complexes, it should not significantly enhance or inhibit the expression of firefly luciferase in FL1181 and FL1182 (Fig. 4A and C). Other antibiotics, due to not affecting DNA supercoiling status in vivo, should not be able to enhance the expression of firefly luciferase. In contrast, they inhibited the expression of firefly luciferase and β-glactosidase in FL1181 and FL1182.

Summary.
Here, using a unique in vivo system, we demonstrated that transient and localized (−) TCDS provided by E. coli RNA polymerase could inhibit the P gyrA at the plasmid and chromosomal levels. We also found that fluoroquinolones, such as ciprofloxacin, were able to substantially increase the expression of the firefly luciferase under the control of the P gyrA in the presence of IPTG. This unique property of TCDS can be effectively used to screen and identify antimicrobial compounds targeting bacterial DNA gyrase.  Plasmid DNA templates. Circular plasmid pZXD133, a derivative of pBR322, was described previously 43 .

Methods
Plasmid pZXD144 was constructed by inserting a 70 bp synthetic oligomer harboring a P gyrA into the BamHI and HindIII sites of pZXD133. In this case, P gyrA is divergently coupled to P T7A1/O4 (Fig. 1). Linear plasmid pZXD150 was described previously 38 .
The expression of β-galactosidase. The expression level of β-galactosidase was measured as described in previous publications 38,49 . Briefly, 100 mL of LB was inoculated with 1 mL of overnight bacterial cell culture at ratio of 1:100 until OD 600 = ~0.2. 100 μL of bacterial cell culture was added to 900 μL of Z-buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , and 50 mM β-mercaptoethanol). Then, 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 minutes, 200 μL of ONPG (4 mg/mL) was added. After another 15 min of incubation at 30 °C, 500 μL of 1 M Na 2 CO 3 was added to stop the reaction. After cell debris was removed by centrifugation at 13,000 rpm for 1 min, the OD 420 and OD 550 values were measured in a Cary 50 spectrophotometer. β-Galactosidase activities (E) were calculated using equation: Luciferase assay. The expression of the firefly luciferase in E. coli were monitored by using the luciferase assay as described in our previous publication 38 .

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. "