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. Author manuscript; available in PMC: 2024 Feb 9.
Published in final edited form as: FEBS Lett. 2019 Nov 14;594(5):791–798. doi: 10.1002/1873-3468.13643

Fis protein forms DNA topological barriers to confine transcription-coupled DNA supercoiling in Escherichia coli

Samantha Dages 1, Xiaoduo Zhi 1, Fenfei Leng 1
PMCID: PMC10857741  NIHMSID: NIHMS1056253  PMID: 31639222

Abstract

Previously, we demonstrated that transcription-coupled DNA supercoiling (TCDS) potently activated or inhibited nearby promoters in Escherichia coli even in the presence of all four DNA topoisomerases, suggesting that DNA topoisomerases are not the only factors regulating TCDS. A different mechanism exists to confine this localized DNA supercoiling. Using an in vivo system containing the TCDS-activated leu-500 promoter (Pleu-500), we find that the nucleoid-associated Fis protein potently inhibits the TCDS-mediated activation of Pleu-500. We also find that deletion of the fis gene significantly enhances TCDS-mediated inhibition of transcription of the rRNA genes purH, yieP, and yrdA in the early log phase. These results suggest that Fis protein forms DNA topological barriers upon binding to its recognition sites, blocks TCDS diffusion, and potently inhibits the TCDS-activated Pleu-500.

Introduction

Transcription can induce localized DNA supercoiling [1–3]. This so-called transcription-coupled DNA supercoiling (TCDS) has been successfully explained by a twin-supercoiled domain model of transcription in which RNA polymerase transcribing along the DNA double helix produces a (+) supercoiling domain in front of the RNA polymerase and a (−) supercoiling domain behind it [4]. Although DNA topoisomerases may relax these two supercoiling domains [5–8], previous studies showed that topoisomerases alone are not sufficient to fully remove TCDS in Escherichia coli [9–11]. In other words, TCDS especially from the strong promoters such as rrn P1, P2 promoters, if not confined, greatly influences the nearby promoters [10,11]. One way for Escherichia coli to control this localized TCDS is forming a DNA topological barrier upstream of these promoters to block its diffusion. In this case, TCDS is confined to a localized region and should not greatly affect the nearby promoters. Additionally, the confined TCDS may self-activate the promoter and meet the fast-growing requirements of E. coli cells at the exponential phase. Nevertheless, if this hypothesis is correct, two key questions need to be answered. Do these DNA topological barriers exist in E. coli? If they exist, what forms DNA topological barriers to confine TCDS? After examining the E. coli genome and related studies [12–14], we found that the E. coli Fis (factor for inversion stimulation) protein is a possible candidate that can form this kind of DNA topological barriers. First, Fis protein has multiple binding sites in the promoter region, usually located in the upstream activator sequence (UAS), to regulate transcription of many genes [12,13]. For instance, the E. coli rrnB P1 promoter has three Fis protein binding sites centered at −71, −102, and −143 [15]. In fact, all seven rrn P1 promoters contains 3–5 Fis protein binding sites in their UAS regions [16,17]. Previous studies showed that Fis protein potently activates rrnB P1 promoter mainly through binding to the promoter proximal site, i.e., site 1 and interacting with the C-terminal domain of the α subunit (αCTD) of RNA polymerase [18]. Intriguingly, binding of Fis protein to sites 2 and 3 further activates transcription from the rrnB P1 promoter [15]. However, the interaction between Fis protein and αCTD cannot explain this activation [17]. Fis protein-mediated DNA topological barriers by forming a DNA microloop [19,20] can explain this activation. In this way, (−) DNA supercoiling is confined within the promoter region and activates the supercoiling-sensitive rrnB P1 promoter [21].

Secondly, one unique feature of Fis protein is the growth phase dependent expression [22]. Stationary phase cells contain very little Fis protein (less than 100 copies per cell) [22]. In contrast, nutritional up-shift of these stationary phase cells dramatically induces the Fis protein expression. At the peak, 50,000–100,000 copies of Fis protein per cell are produced [22]. Afterwards, Fis protein synthesis is turned off and the cellular Fis protein level continuously decreases due to cell division [22]. This growth phase dependent expression pattern of Fis protein is coincident with the transcription pattern of those genes involved in E. coli cell growth [12,16] and therefore the level of TCDS. Since the main function of Fis protein is to regulate the supercoiling level of E. coli chromosome [23–29], it has the potential to form DNA topological barriers to block TCDS diffusion. Third, Fis protein is one of the most abundant nucleoid-associated proteins (NAPs) [30]. This small dimeric DNA-binding protein binds to a type of 15 bp degenerate DNA sequences 5’-GN13C-3’ with 5–7 AT base pairs in the center [12,13,31] by recognizing the narrow minor groove of the central AT base pairs and further compresses the minor groove upon binding [32,33]. Consequently, the DNA bends ~65° toward the Fis protein [32]. Interestingly, EM and AFM studies showed that Fis protein preferentially binds to the crossovers of supercoiled DNA molecules and stabilizes branched plectonemes or DNA loops [34,35]. These compact nucleoprotein structures are similar to the LacI-mediated DNA topological barriers that efficiently block DNA supercoiling diffusion [36–38]. Furthermore, a recent study showed that Fis protein was able to help form transient and dynamic chromosome domain boundaries/topological barriers to block supercoiling diffusion [39]. All these results support the hypothesis that Fis protein can form topological barriers along the bacterial genome and block supercoiling diffusion. In this article, utilizing a previously established in vivo system in which the supercoiling-sensitive promoter Pleu-500 can be activated by TCDS [10], we examine how Fis protein regulates the TCDS-activated Pleu-500. Our results demonstrate that Fis protein potently inhibited the TCDS-activated Pleu-500. Simultaneously, Fis protein also self-activated transcription from the rrnB P1, P2 promoters. Our results are consistent with a model in which Fis protein forms DNA topological barriers, blocks TCDS diffusion, and potently inhibits the activation of Pleu-500. Likewise, TCDS generated from the strong rrnB P1, P2 promoters bounced back and self-activated these two promoters.

Materials and methods

Materials.

The following materials/products were purchased from different companies: restriction enzymes and T4 DNA ligase, New England Biolabs; pfu DNA polymerase, Stratagene; synthetic oligonucleotides, Eurofins Genomics; QIAprep Spin Miniprep Kit, QIAquick Gel Extraction Kit, RNeasy Mini Kit, and QIAquick Nucleotide Removal Kit, QIAGEN; ThermoScript RT-PCR System plus Platinum® Taq DNA polymerase, Invitrogen; Power SYBR Green PCR Master Mix, Applied Biosystems; Luciferase Assay System, Promega; and Isopropyl-β-D-thiogalactopyranoside (IPTG), Gold Biotechnology.

Plasmids.

Plasmids pZXD and pZXD-Fis were derived from plasmid pZXD133 [10]. pZXD was constructed by inserting a 184 bp synthetic oligomer containing the IPTG inducible rrnB P1 and P2 promoters (Fig. 1A) between EcoRI and XhoI sites of pZXD133. pZXD-Fis was made by inserting a 296 bp DNA fragment that contains 3 Fis protein-binding sites and the IPTG inducible rrnB P1 and P2 promoters (Fig. 1B) between EcoRI and XhoI sites of pZXD133.

Figure 1. (A) and (B) The supercoiling-sensitive promoter Plue-500 divergently coupled to the IPTG-inducible rrnB P1 and P2 promoters.

Figure 1.

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Divergently coupled promoters Pleu-500 (green horizonal oval) and the IPTG-inducible rrnB P1 and P2 promoters (blue vertical oval) were used to control the expression of firefly luciferase (luc) and β-galactosidase (lacZ), respectively. The open vertical oval represents the lac O1 operator. Red rectangle represents Fis protein-binding sites. The DNA sequence of the coupled promoters Pleu-500 (highlighted in green) and the IPTG-inducible rrnB P1 and P2 promoters (highlighted in blue) are shown. Boxed are −10 and −35 regions of Pleu-500 and PT7A1/O4. Construct A does not have Fis protein-binding sites. The three Fis protein-binding sites between Pleu-500 and the IPTG-inducible rrnB P1 and P2 promoters in construct B mimic the E. coli rrnB operon. (C) and (D) Fis protein-binding sites dramatically affect rrnB P1, P2 promoters and the divergently coupled Pleu-500 at the chromosome level during the E. coli exponential growth phase. Overnight cultures of E. coli strains FL1261 (black dots and lines) and FL1263 (red dots and lines) were diluted 100-fold and grown in the presence of various amounts of IPTG to an OD600 of 0.5, and assayed for β-galactosidase (Miller units) and luciferase activities. Light emitted by luciferase was monitored in a luminometer. Raw light units (RLU) were plotted against the IPTG concentration added to the cell cultures.

E. coli strains.

Escherichia coli strain MG1655 [F−, λ−, rph-I] was obtained from the Coli Genetic Stock Collection/E. coli Genetic Resource Center (CGSC) at Yale University. E. coli strain FL1261 (MG1655(DE3)ΔlacZ attTn7::rrnBP1,P2/lacO1lacZ-Pleu-500luc) was described previously [10]. E. coli strain FL1263 (MG1655(DE3)ΔlacZ attTn7::rrnBP1,P2/lacO1lacZ-Fis-Pleu-500luc) was constructed by using a Tn7-based site-specific recombination system [40], as described before. The difference between FL1261 and 1263 is the DNA sequence between Pleu-500 and the IPTG-inducible P1, P2 promoters (Fig. 1). FL1263 carries three Fis protein-binding sites derived from rrnB operon. E. coli strain MG1655Δfis was constructed using the λ Red recombination system [41]. Fig. S1 shows the growth curves of MG1655 and MG1655Δfis in LB, which is consistent with previously published results [23].

The expression of β-galactosidase.

The expression level of β-galactosidase was measured by Miller’s assay as described previously [10,42]. Briefly, 100 mL of LB was inoculated with 1 mL of overnight bacterial cell culture until OD600 reached ~0.5. Then 100 μL of fresh bacterial cell culture was mixed with 900 μL of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM β-mercaptoethanol). Cells were lysed with 60 μL of chloroform plus 30 μL of 0.1% SDS. After 5 min incubation at 30 °C, 200 μL of 4 mg/mL ONPG was added to the cell lysates. After an additional 15 min incubation at 30 °C, reactions were stopped by the addition of 500 μL of 1 M Na2CO3. Cell debris were removed by centrifugation at 13,000 rpm for 1 min. OD420 and OD550 were measured in a Cary 50 spectrophotometer. β-Galactosidase activities (E) were calculated using the following equation:

E=1000×OD420–1.75×OD550t×v×OD600 (1)

where t and v represent reaction time and cell culture volume, respectively.

Luciferase Assay.

Luciferase Assay to measure the expression of the luciferase gene in E. coli strains was described previously [10].

RNA isolation, cDNA synthesis, and real-time polymerase chain reaction (PCR).

Total RNA was isolated from E. coli cells using QIAGEN RNeasy Kit as described previously [10]. cDNA synthesis was also described previously [10]. Real-time PCR experiments were carried out using a Biorad MiniOpticon Real-time PCR instrument. Briefly, a 20 μL reaction containing 1 μL cDNA, 0.5 μM of each primer, and 10 μL of Power SYBR Green PCR Master Mix (2×) was incubated at 95 °C for 10 min and continued for 40 cycles at 95 °C, 15 s and 60 °C, 1 min. The Cq values were calculated from exponential phase of each PCR reaction. Table S1 summaries primers used in the RT-PCR reactions. Primers are listed in Table S1.

Results and discussion

We have previously established an in vivo system to study how TCDS affects the divergently-coupled, supercoiling-sensitive promoters, i.e., Pleu-500 and PgyrA [10,11]. Our results showed that this transient, localized, and powerful supercoiling force greatly stimulated Pleu500 [10] and potently inhibited PgyrA [11]. In this paper, we decided to examine how Fis protein, a nucleoid associated protein (NAP) regulates TCDS and TCDS’ effects on the divergently coupled Pleu-500. For this purpose, we constructed two E. coli strains, FL1261 and FL1263. As described in our previous publication [10], FL1261, a derivative of MG1655 carries a ~5 kb DNA fragment at the attTn7 site containing the IPTG-inducible E. coli ribosomal rrnB P1, P2 promoters divergently coupled to Pleu-500 with luc and lacZ genes (Fig. 1A). FL1263 is identical to FL1261 except it also contains three Fis protein-binding sites, derived from the upper stream sequence of rrnB P1, P2 promoters, between the IPTG-inducible ribosomal rrnB P1, P2 promoters and Pleu-500 (Fig. 1B). At the cell growth exponential phase, we added IPTG into the cell culture to induce the expression of β-galactosidase controlled by the rrnB P1, P2 promoters and measure the activities of Pleu-500 using the luciferase assay. As expected, TCDS potently stimulated Pleu-500 for both E. coli strains (Fig. 1C and D). However, some differences are observed. TCDS was only able to activate Pleu-500 2.7-fold for FL1263 that contains three Fis protein-binding sites between the IPTG-inducible ribosomal rrnB P1, P2 promoters and Pleu-500. In contrast, TCDS was able to activate Pleu-500 17.5-fold for FL1261 that does not carry these Fis protein-binding sites (Fig. 1D). Since the distance between the −35 region of Pleu-500 and the nearest Fis protein-binding site is 72 bp (Fig. 1B) and too far for Fis protein to block E. coli RNA polymerase from binding to Pleu-500, the most straightforward explanation is that Fis protein serves as a DNA topological barrier to block TCDS from the rrnB P1, P2 promoters. In this way, the supercoiling force that the Pleu-500 experienced in FL1263 was significantly reduced. As a result, the Pleu-500 activity in FL1263 was much lower than that in FL1261. We also observed that the expression level of β-galactosidase between these two E. coli strains are different. The expression level of β-galactosidase for FL1263 (with Fis protein-binding sites) is two times more than that for FL1261 (without Fis protein-binding sites). This is consistent with previous studies demonstrating that the Fis protein-activated transcription stems from the interaction between Fis protein and the αCTD of RNA polymerase [17] and also from Fis protein stabilizing the interaction between RNA polymerase and the rrnB P1 promoter [43]. It is also possible that the Fis protein-mediated DNA topological barrier may not only block TCDS from the rrnB P1, P2 promoters, but also keep the supercoiling on the rrnB P1, P2 side and further activate these two promoters.

Since the chromosome location and the genetic elements around the attTn7 site may have an impact on TCDS and Fis proterin’s effect on TCDS, we cloned these two ~5 kb DNA fragments at the attTn7 site containing the IPTG-inducible E. coli ribosomal rrnB P1, P2 promoters divergently coupled to Pleu-500 with luc and lacZ genes into a pBR322-derived plasmid [10] to yield two plasmids, pZXD (without Fis protein-binding sites) and pZXD-Fis (with Fis protein-binding sites between the IPTG-inducible ribosomal rrnB P1, P2 promoters and Pleu-500) (Fig. 2A and B). We then performed the luciferase assays and Miller’s assays using E. coli strains MG1655(DE3)ΔlacZ containing these two plasmids in the absence and presence of IPTG induction during the cell growth exponential phase. Our results are summarized in Fig. 2C and D. Apparently, Fis protein-binding sites significantly affected the expression profiles of E. coli strains MG1655(DE3)ΔlacZ containing these two plasmids. In the absence of IPTG, the IPTG-inducible rrnB P1, P2 promoters in MG1655(DE3)ΔlacZ had substantial basal or leaking transcription and expression of β-galactosidase, which is consistent with our previously published results [37]. The basal transcription of β-galactosidase was able to activate Pleu-500 and resulted in the substantial expression of the firefly luciferase (Fig. 2D). However, the expression profiles of β-galactosidase and luciferase for MG1655(DE3)ΔlacZ containing these two plasmids are quite different. MG1655(DE3)ΔlacZ carrying pZXD had a much lower expression level of β-galactosidase compared to MG1655(DE3)ΔlacZ carrying pZXD-Fis. In contrast, the expression level of luciferase of MG1655(DE3)ΔlacZ carrying pZXD was significantly higher than that of MG1655(DE3)ΔlacZ carrying pZXD-Fis. Similar results were also observed for MG1655(DE3)ΔlacZ containing these two plasmids after IPTG induction (Fig. 2C and D). As expected, IPTG substantially induced the expression of β-galactosidase and firefly luciferase for both plasmids. MG1655(DE3)ΔlacZ carrying pZXD had a lower expression level of β-galactosidase compared to MG1655(DE3)ΔlacZ carrying pZXD-Fis and, however, had a higher expression level of the firefly luciferase than that of MG1655(DE3)ΔlacZ carrying pZXD-Fis. These results demonstrate that at the plasmid level Fis protein upon binding to its binding sites is able to form topological barriers to block TCDS from the rrnB P1, P2 promoters and therefore inhibit the TCDS-induced activation of Pleu-500. Intriguingly, the effects of Fis on plasmids were greatly amplified compared to its effects on the chromosome (compare Fig. 1 to Fig. 2).

Figure 2. Effects of Fis protein-binding sites on the rrnB P1, P2 promoters and the divergently coupled Pleu-500 at the plasmid level during the E. coli exponential growth phase.

Figure 2.

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Plasmid maps of pZXD (A) and pZXD-Fis (B) are shown. Plasmid pZXD-Fis contains three Fis-binding sites as described in Fig. 1B. Miller’s assays and luciferase assays are described in Materials and Methods, and also in Fig. 1. (C) and (D) Plasmids were transformed into E. coli strain MG16155(DE3)ΔlacZ. (E) and (F) Plasmids were transformed into E. coli strain MG16155(DE3)ΔfisΔlacZ where the fis gene was deleted from the E. coli chromosome.

We also transformed these two plasmids into E. coli strain MG1655(DE3)ΔlacZΔfis in which the fis gene was deleted from the chromosome using λ Red recombination system [41]. We then performed the luciferase assays and Miller’s assays using E. coli strains MG1655(DE3)ΔlacZΔfis containing these two plasmids in the absence and presence of IPTG induction during the cell growth exponential phase. Fig. 2E and F show our results. As expected, TCDS potently activated Pleu-500 and stimulated the expression of firefly luciferase (Fig. 2F). However, there is not much differences between E. coli strains MG1655(DE3)ΔlacZΔfis carrying pZXD and pZXD-Fis. In other words, the effects of Fis protein on the expression of β-galactosidase and firefly luciferase were almost completely eliminated (Fig 2E and F), suggesting that Fis protein has a fundamental influence on TCDS.

Previously we analyzed the seven rrn operons of E. coli and found that three genes, i.e., purH, yieP, and yrdA are divergently coupled to the P1, P2 promoters of rrnE, rrnC, and rrnD operons, respectively [10,44]. However, only one σ70 promoter, the purH promoter is divergently coupled to the P1, P2 promoters of the rrnE operon (Fig. 3A). No σ70 promoters are associated with yieP and yrdA (only transcription starting sites (TSSs) are found) (Fig. 3A). Due to the fact that the rRNA promoters are very active in the early log phase and greatly depressed in the stationary phase, TCDS from rRNA transcription greatly inhibited the transcription of purH, yieP, and yrdA in the early log phase compared to the stationary phase [10]. Interestingly, all seven rrn operons carry tandem copies of the Fis protein binding sites on the upstream regions of the strong P1, P2 promoters [17]. If our hypothesis, in which Fis protein serves as DNA topological barriers to block TCDS, is correct, deletion of fis gene from the chromosome should further depresses the transcription of purH, yieP, and yrdA in the early log phase. Indeed, our RT-PCR results clearly showed that the transcription level of purH, yieP, and yrdA in the early log phase in MG1655Δfis was significantly lower than that in MG1655 (Fig. 3B). In contrast, their transcription level was enhanced in the stationary phase. These results strongly support the hypothesis that Fis protein serves as a DNA topological barrier to prevent TCDS from activating or inhibiting other nearby promoters (Fig. 4). In this scenario, the Fis protein-mediated topological barriers give DNA topoisomerases more time to remove the excess, harmful supercoiling.

Figure 3. Real-time RT-PCR analyses of transcription levels of purH, yrdA, and yieP in the early log phase and stationary phase for MG1655 and MG1655Δfis.

Figure 3.

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RNA samples were purified from the early log phase (OD600=~0.5; L, red) and the stationary phase (the overnight culture; S, green). The standard deviation was calculated according to three independent experiments.

Figure 4.

Figure 4.

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Fis protein-mediated DNA topological barrier blocks transcription-coupled DNA supercoiling (TCDS) diffusion from RNA polymerase 1 (RNAP1, blue oval), which significantly decreases the effect of TCDS on the divergently coupled transcription by RNAP2 (green oval). Arrows indicate the transcription directions by RNAP1 and RNAP2.

In summary, using genetic and biochemical techniques, we demonstrated that Fis protein strongly inhibits TCDS-activated Pleu-500 on the chromosome and plasmids. We also found that the deletion of the fis gene from the chromosome significantly enhanced the TCDS-mediated inhibition of the transcription of purH, yieP, and yrdA in the early log phase. Our results are consistent with a hypothesis that Fis protein upon binding to the Fis protein-binding sites forms topological barriers and blocks supercoiling (TCDS) diffusion. As a result, these Fis protein-based DNA topological barriers confine TCDS to localized regions and reduce the effect of TCDS on the divergently-coupled promoter and/or transcription.

Supplementary Material

Supp info

Acknowledgments and Funding

This work was supported by grants 1R15GM109254-01A1 and 1R21AI125973-01A1 from the National Institutes of Health (to F.L.).

Footnotes

Competing Interests

The authors declare no competing interests.

Additional Information

Supplementary information accompanies this paper at FEBS Letters web site.

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