# The Escherichia coli colibactin resistance protein ClbS is a novel DNA binding protein that protects DNA from nucleolytic degradation

Katja Molan, Zdravko Podlesek, Vesna Hodnik, Matej Butala, Eric Oswald

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Katja Molan, Zdravko Podlesek, Vesna Hodnik, Matej Butala, Eric Oswald. The Escherichia coli colibactin resistance protein ClbS is a novel DNA binding protein that protects DNA from nucleolytic degradation. DNA Repair, 2019, 79, pp.50-54. 10.1016/j.dnarep.2019.05.003. hal-02618217

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Submitted on 25 May 2020

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<figure><img src="image_1.png" /></figure>

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<table><caption>**Table 1**
Bacterial strains and plasmids used in this study.</caption><thead><tr><th>Bacterial strain/plasmid</th><th>Description</th><th>Source</th></tr></thead><tbody><tr><td>DH5α (DE3)</td><td>Expression host, harboring the lambda DE3.</td><td>Thermo Fisher Scientific</td></tr><tr><td><i>E. coli</i> BL21(DE3)</td><td>Expression host, harboring the lambda DE3; Cm<sup>R</sup>.</td><td>Thermo Fisher Scientific</td></tr><tr><td>BAC pks<sup>+</sup></td><td>The source of the <i>clbS</i> gene.</td><td>(2)</td></tr><tr><td>pUSP4</td><td>The source of the <i>usp</i> gene; Ap<sup>R</sup>.</td><td>(17)</td></tr><tr><td>pET8c</td><td><i>E. coli</i> T7 expression vector, containing an N-terminal hexahistidine tag (His<sub>6</sub>) and thrombin cleavage site.</td><td>Novagen</td></tr><tr><td>pCLB-his</td><td>pET8c with <i>clbS</i> gene.</td><td>This study</td></tr><tr><td>pLysS</td><td>pLys with removed lysozyme gene; Cm<sup>R</sup>.</td><td>Thermo Fisher Scientific</td></tr><tr><td>pLCUBI</td><td>pLysS with <i>clbS</i> and <i>usp</i> genes; Cm<sup>R</sup> and Ap<sup>R</sup>.</td><td>This study</td></tr><tr><td>pLUBI</td><td>pLysS with <i>usp</i> gene; Cm<sup>R</sup> and Ap<sup>R</sup>.</td><td>This study</td></tr><tr><td>pSC201</td><td>Plasmid with <i>recA::gfp</i> fusion; Kn<sup>R</sup>.</td><td>(18)</td></tr></tbody></table>

GAG-3') and ClbS-R (5'-TTACGCGTTATTCTGCAAGACATTTCTGCAG-3') primers (Sigma-Aldrich) at 55 °C annealing temperature. The PCR product, digested with BamHI and MluI, was cloned into the expression vector pET8c, in a fusion with a thrombin-cleavable N-terminal hexahistidine tag (His<sub>6</sub>-tag). This plasmid, designated pCLB-his, was subsequently used for ClbS protein isolation and for the construction of vectors, used for in vivo studies. Sequencing of the inserted clbS fragment was performed to check for potential PCR introduced mutations. For the construction of vectors, the BamHI and EcoRV digested and blunted fragment of the low copy number pLysS plasmid was employed. This backbone fragment contained only the replication region and the chloramphenicol resistance gene, with the T7 lysozyme gene removed. The source of the <i>usp</i> gene for subcloning into the pLysS fragment was plasmid pUSP4 [17]. Two plasmids were thus constructed, pLUBI, carrying the <i>usp</i> gene but without its immunity genes, and pLCUBI harbouring <i>usp</i> and <i>clbS</i>, and were transformed into <i>E. coli</i> BL21 (DE3). Both genes were controlled by independent T7 polymerase promoters. To follow the SOS response, plasmid pSC201 with a <i>recA::gfp</i> fusion was used [18], transformed into either <i>E. coli</i> BL21 (DE3) with pLCUBI, or <i>E. coli</i> BL21 (DE3) with pLUBI.

## 2.2. Expression, Isolation, purification and detection of the ClbS protein

ClbS was expressed in *E. coli* strain BL21 (DE3) pCLB-his. An overnight culture was used to inoculate liquid Luria Bertani medium supplemented with 25 µg/mL chloramphenicol and grown at 37 °C to an OD<sub>600</sub> 0.5 when ClbS production was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.8 mM. The culture was subsequently grown for an additional 4 h when biomass was collected by 10 min centrifugation at 4.000 × g. His<sub>6</sub>-tagged ClbS was purified by Ni-NTA metal affinity chromatography (Qiagen) and stored in elution buffer (50 mM NaH<sub>2</sub>PO<sub>4</sub>, 300 mM NaCl, 250 mM imidazole, pH 8) at 4 °C. Further, overnight dialysis was performed against a final buffer (50 mM NaH<sub>2</sub>PO<sub>4</sub>-H<sub>2</sub>O, 200 mM NaCl, 20 mM β-mercaptoethanol, pH 8) at 4 °C. The concentration of the purified ClbS protein was determined using a NanoDrop 1000 (Thermo Fisher Scientific) with the extinction coefficient at 280 nm of 37530 M<sup>-1</sup> cm<sup>-1</sup> and by Pierce BCA protein assay kit, using Bovine Serum Albumin (BSA) (Sigma-Aldrich) as the standard. Additionally, the His<sub>6</sub>-tag was removed from His<sub>6</sub>-ClbS with the Thrombin cleavage capture kit (Novagen) and the buffer exchanged by overnight dialysis as described above. To assay the removal of the affinity tag, the thrombin-digested or undigested His<sub>6</sub>-ClbS samples were resolved on a 12% acrylamide gel (Fig. S1).

## 2.3. Electrophoretic mobility shift assays (EMSA)

Various concentrations of ClbS carrying or lacking the affinity His<sub>6</sub>-tag (0.15 µM – 2.4 µM) and 60 ng/µL of DNA (Table 2) were used to establish the nucleic acid-binding ability of ClbS. ClbS was incubated with DNA in 1 X FastDigest Green Buffer (Fermentas) at 37 °C for

15 min, prior to the analysis of the protein-DNA complexes by EMSA analysis on 1.5–2 % agarose gels. As a negative control BSA was included at a final concentration of 1.6 µM. Additionally, mixtures of pUC19/EcoRI (60 ng/µL) and ClbS (0.15 µM – 2.4 µM) were pre-incubated at 37 °C for 5–15 minutes when colicin E7 (ColE7), isolated from *E. coli* [19] or commercial DNase I (Sigma-Aldrich) were added in final concentrations 0.42 mg/ml and 0.5 mg/mL, respectively, followed by an additional 30 min incubation. As a control, DNA without ClbS was incubated with ColE7 or commercial DNase I. Results were analyzed employing 2% agarose gels.

## 2.4. Surface plasmon resonance assays (SPR)

Measurements were performed at 25 °C on a Biacore T100 (GE Healthcare, UK) at the Infrastructural Centre for Analysis of Molecular Interactions (University of Ljubljana, Slovenia). To prepare biotinylated DNA for immobilization on the streptavidin sensor chip, plasmid pJBPL-A was used as a matrix DNA to amplify the 403 bp long DNA sequence (from *Salmonella bongori*). Annealing temperature of 55 °C was used to amplify with primers Bong-biotin (5'-CATAAAACCTCGTGTA TGGTGCGC-3 and Bong-R (5'-GAATTCCATTTTACGACATCCGAATAT AAATTAAACGAA-3') (Integrated DNA Technology). Approximately 50 response units (RU) of the 3'-biotinylated 403 bp long PCR product were immobilized on the second flow cell of the streptavidin (SA) sensor chip (GE Healthcare) in SPR buffer (20 mM Tris (pH 7.4), 140 mM NaCl, 2 mM DTT, 0.005% surfactant P20 and 0.1 mg/ml BSA). The first flow cell was left empty and used to control any non-specific binding to the sensor surface. The interaction between ClbS and chip-immobilized DNA was studied by injecting various concentrations of ClbS in SPR buffer. Injections of ClbS over the immobilized DNA fragment were performed at a flow rate of 10 µl min<sup>-1</sup> for 2 min with dsDNA and 4 min with ssDNA. The sensor chip with bound DNA was regenerated by injection of SPR buffer containing 0.03% SDS for 15 s at a flow rate of 30 µl min<sup>-1</sup>. Further, chip-immobilized dsDNA denaturation was applied with 2 pulses of 50 mM NaOH for 30 s to obtain ssDNA. The response after NaOH treatment dropped for approximately 20 RU, indicating that one DNA chain was removed. Injections of various concentrations of ClbS in SPR buffer was applied at a flow rate of 10 µl min<sup>-1</sup> for 4 min. SPR experiments were performed in triplicate and representative experiments are shown. The data were analyzed with the Biacore T100 Evaluation Software and equilibrium dissociation constants (K<sub>D</sub>) were determined by fitting the data to the steady state affinity model.

## 2.5. Quenching of intrinsic ClbS tryptophan fluorescence by ss- and dsDNA

Intrinsic tryptophan fluorescence of ClbS (40 µg/mL in 50 mM phosphate buffer, 200 mM NaCl, β-mercaptoethanol, pH 8.0) was measured. DNA used for titrations are presented in Table 2. After adding DNA, a 10 s incubation followed when measurements were made in a constantly stirred 1 cm path length quartz cuvette with a scan
<table><caption>**Table 2**
DNA employed in this study.</caption><thead><tr><th>DNA</th><th>Length<br>(bp or bases)</th><th>Application</th><th>Reference</th></tr></thead><tbody><tr><td>pUC19</td><td>2686</td><td>EMSA</td><td>Fermentas</td></tr><tr><td>pJBPL-A</td><td>3377</td><td>Matrix DNA for PCR reaction.</td><td>This laboratory</td></tr><tr><td>PCR amplicon</td><td>403</td><td>SPR (ds- and ssDNA), EMSA,<br>Trp fluorescence quenching</td><td>This study</td></tr><tr><td>ds- and ssDNA<br>phage genome M13</td><td>6407</td><td>EMSA,<br>Trp fluorescence quenching</td><td>[20]</td></tr></tbody></table>

rate of 250 nm/min (37 °C, excitation and emission slits at 5 nm each). The fluorescence emission spectra were recorded in the wavelength range of 300–450 nm after excitation at 295 nm, specifically monitoring the tryptophan fluorescence emission. Fluorescent emissions of the buffer titrated with DNA (background intensities) were subtracted.

## 2.6. Growth of *E. coli* clbS<sup>+</sup> *usp<sup>+</sup>* and *E. coli* *usp<sup>+</sup>* strains and cell morphology analysis

The two *E. coli* BL21 (DE3) strains were cultivated at 37 °C in liquid BHI medium with ampicillin and chloramphenicol at final concentrations 100 and 20 µg/ml, respectively. To suppress basal level expression of the T7 polymerase promoter, glucose was added at a final concentration of 0.4%. When cultures reached OD<sub>600</sub> 0.5, protein synthesis was induced by addition of IPTG at final concentration 0.8 mM. One ml of both cultures was sampled before induction and each hour following induction (at least three hours). The OD<sub>600</sub> was measured and plotted in time.

Prior to microscopy, the *E. coli* BL21 (DE3) clbS<sup>+</sup> *usp<sup>+</sup>* and *E. coli* *usp<sup>+</sup>* strains were transformed with a plasmid carrying a *recA::gfp* gene fusion and cultured as described above in media supplemented with 30 µg/ml kanamycin. Both cultures were sampled before and every hour following induction. Cells were washed twice with NaCl and attached to glass slides coated with 0.1% (w/v) poly-L-lysine (Sigma). Cell morphology was observed under light and *recA::gfp* expression under fluorescence microscopy, using a Zeiss AxioImager Z1 microscope equipped with a HRC digital camera.

# 3. Results and discussion

## 3.1. ClbS binds ss- and dsDNA in a non-specific manner

Initially, EMSA was applied and showed that ClbS causes circular and linear DNA retardation (Fig. 1A) and, that it interacts with ss- or dsDNAs of various lengths and source (Table 2, Fig. S2). Thus, EMSA experiments implied that ClbS binds DNA in a non-specific manner. It is of note that ClbS binding activity was not affected by the His<sub>6</sub> tag (Fig. S3). SPR spectrometry measurements of ClbS binding to ssDNA (Fig. 1B) or dsDNA (Fig. 1C) revealed an approximately 40-times higher affinity of ClbS for ssDNA exhibiting an apparent equilibrium dissociation constant (K<sub>D</sub>) of 24.5 ± 1.2 nM compared to dsDNA exhibiting a K<sub>D</sub> of 936.8 ± 12.3 nM (Fig. 1D). Additionally, to confirm that ClbS interacts with DNA we assayed the tryptophan fluorescence spectra of ClbS, which carries five tryptophan residues, in the presence or in the absence of DNA. We observed quenching of intrinsic ClbS tryptophan fluorescence by various ss- and dsDNAs. The most pronounced decrease in intensity of emission maximum was observed upon titration with ssDNA (Fig. 1E). Our results implied that the five ClbS tryptophan residues behave as two populations of fluorophores, one accessible to quenchers and the other buried in the protein structure [21], presented as downward curvature in the Stern-Volmer plot (Fig. 1F).

## 3.2. ClbS protects DNA from deoxyribonucleases

DNA protection by ClbS was examined by measuring growth of the *E. coli* strain carrying a plasmid encoded *usp* gene and its cognate immunity genes replaced with clbS, in comparison to a strain harbouring only the *usp* gene (Table 1). Expression of *usp* and clbS was controlled by the T7 polymerase promoter. Following IPTG induction of *usp* and clbS the strain encoding only *usp* (lacking its immunity genes or clbS), exhibited dramatically reduced growth in comparison to the strain with both *usp* and clbS (Fig. 2A). DNA damage induces the SOS response involved in DNA repair. The principal regulators of the SOS response are the LexA repressor and RecA activator. Upon DNA damage, RecA is activated by binding to exposed ssDNA which stimulates LexA autocleavage, derepressing SOS genes. SOS induction inhibits cell division, resulting in cell filamentation [22]. Light microscopy of IPTG-induced cells expressing *usp* revealed pronounced filamentation (Fig. 2B), while typical cell shape and size were detected among *E. coli* expressing both *usp* and clbS genes (Fig. 2C). The "two examined strains also carried a plasmid encoded *recA::gfp*, a DNA damage SOS reporter gene fusion. Fluorescent microscopy revealed that *usp<sup>+</sup>* cells without clbS, additionally exhibited elevated expression of *recA::gfp* (Fig. 2D) when compared to the *usp<sup>+</sup>* clbS<sup>+</sup> cells (Fig. 2E). Filamentation and elevated expression of *recA* are hallmarks of induction of the DNA damage inducible SOS response. Thus, typical cell shape and size, as well as basal level expression of *recA* in *usp<sup>+</sup>* clbS<sup>+</sup> cells indicates, that ClbS protects cells from Usp nuclease activity.

We subsequently hypothesized that binding of ClbS could provide DNA protection against other deoxyribonucleases. Co-incubation of DNA and ClbS protected plasmid DNA against DNase I (Fig. 2F) and the ColE7 nuclease (Fig. 2G) in a concentration dependent manner, confirming that ClbS provides protection against various nucleases by presumably interacting non-specifically with DNA. ClbS binding to DNA is evident as DNA retardation (Fig. 2G, and F). Other examples of protective bacterial proteins have been characterized previously namely, two *E. coli* nucleoid associated proteins, the multifunctional Dps and CbpA, that non-specifically bind DNA, to drive nucleoid compactation and DNA protection [23–26]. Further, the α/β type small acid-soluble spore proteins that are synthesized during sporulation, bind DNA changing its conformation to an A-like helix, that is more resilient against damage [27,28].

In conclusion, our results imply that ClbS, besides directly interacting with colibactin is also a novel DNA binding protein. Thus, beyond colibactin, ClbS also protects DNA against degradation/genotoxic activity presumably providing a selective advantage for bacterial strains encountering stressful conditions also within the host.

## Conflict of interest

The authors have no conflict of interest.

## Footnotes

Data accessibility available via Mendeley Data: https://data.mendeley.com/datasets/4y3wpkrfzc/draft?d=
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** ClbS is a DNA binding protein. (A) EMSA of ClbS interacting with the DNA. M designates marker (bp). Lane 1 contains pUC19 plasmid DNA, lane 2 pUC19 with BSA and lane 3 pUC19 with ClbS. Lane 4 contains pUC19 linearized with EcoRI, lane 5 pUC19/EcoRI with BSA and lane 6 pUC19/EcoRI with ClbS. (B) SPR sensorgrams showing the interaction of ClbS with 403 bp long chip-immobilized ssDNA or (C) dsDNA. (D) The apparent equilibrium dissociation constant ($K_D$) values of ClbS interacting with ssDNA, red or with dsDNA, black. The average $K_D$S and standard deviations were determined from three titrations of each protein. (E) Quenching of intrinsic tryptophan fluorescence of ClbS titrated with ssDNA. The legend denotes ssDNA concentrations preincubated with ClbS. (F) Stern-Volmer plot of tryptophan quenching within ClbS due to interaction with ssDNA or dsDNA.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** ClbS protects DNA from deoxyribonucleases. (A) Growth curves of *E. coli* usp⁺clbS⁺ (black squares) and *E. coli* usp⁺ strains (white squares). Induction of proteins at time 0 h. The experiment was performed seven times, and the means ± standard errors of the means (error bars) are shown. (B) Light and (D) fluorescent microscopy of the *E. coli* usp⁺ strain following 3 h - induction. (C) Light and (E) fluorescent microscopy of the *E. coli* usp⁺clbS⁺ strain following 3 h - induction. (F and G) ClbS protects DNA against DNase degradation. (F) M designates marker (bp). Lane 1 contains pUC19/EcoRI plasmid DNA, lane 2 DNA with DNase I (0.5 mg/mL), lanes 3–7 DNA with ClbS (2.4-0.15µM) and DNase I. (G) M designates marker (bp). Lane 1 contains pUC19/EcoRI DNA, lane 2 DNA with ColE7 (0.42 mg/ml) and lanes 3–6 DNA with ClbS (2.4-0.15µM) and ColE7.</figcaption></figure>

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## Acknowledgements

We thank dr. Miloš Vittori for assistance with microscopy and prof.

dr. Kristina Sepčič with interpretation of results obtained from intrinsic ClbS tryptophan fluorescence quenching. This work was supported by grant P1-0198 and J1-8150 from the Slovene Research Agency. Katja Molan is a recipient of a PhD grant from the Slovene Research Agency.
# Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.dnarep.2019.05.003.

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