Sequential XylS-CTD Binding to the Pm Promoter Induces DNA Bending Prior to Activation

Bacterial strains, culture media, and plasmids.The bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains were grown at 30°C in Luria-Bertani medium supplemented, when required, with 100 μg/ml ampicillin, 25 μg/ml kanamycin, or 20 μg/ml rifampin. Escherichia coli BL21(DE3) was grown on 2×YT medium (1.6% [wt/vol] Bacto tryptone, 1% [wt/vol] yeast extract, and 0.5% [wt/vol] sodium chloride) for protein production.

Cloning, overexpression, and purification of His-tagged XylS derivatives.The 968-bp DNA fragment covering wild-type XylS gene was PCR amplified from the TOL plasmid DNA, digested with the NdeI and XhoI enzymes, and cloned into the pET16b vector (Novagen). The resulting clones were sequenced to confirm XylS sequence. The 400-bp DNA fragment covering the XylS C-terminal domain (XylS residues 196 to 321) was PCR amplified from the TOL plasmid DNA, digested with NdeI and XhoI enzymes, and cloned into the pET16b vector (Novagen). The resulting clones were sequenced to confirm XylS-CTD sequence. To purify the truncated derivative XylS-CTD protein (XylS C-terminal domain), freshly transformed BL21(DE3) cells harboring pET-XylS-C plasmid were grown in 500 ml 2× YT medium (53) at 30°C until turbidity at 660 nm reached 0.5. Culture was then transferred to 16°C and incubated with 0.1 mM isopropyl-beta-d-thiogalactopyranoside (IPTG) for 3 to 4 h, and cells were pelleted and frozen. The cell pellet was resuspended in 50 ml of lysis buffer (20 mM sodium phosphate, pH 7.5, 300 mM NaCl, 0.1 mM EDTA, 2.5 mM 2-mercaptoethanol, 10% [vol/vol] glycerol, 10 mM imidazole, and 1 mM Complete [Roche] protease inhibitor cocktail) and disrupted in a French pressure cell. The crude extract was centrifuged at 30,000 × g for 1 h, filtered through a 0.45-μm pore-size filter and loaded onto a 5 ml Ni-agarose column (Amersham Biosciences) preequilibrated with buffer A-XylS-C (lysis buffer without the Complete cocktail). The column was washed with buffer A-XylS-C until nonspecifically bound material had been removed. A 40-ml imidazole gradient in buffer A-XylS-C (from 0 to 1 M) was then applied. XylS-CTD eluted at about 500 mM imidazole. Eluted fractions containing XylS-CTD protein were dialyzed against storage buffer (20 mM sodium phosphate, pH 7.5, 300 mM NaCl, 2 mM dithiothreitol [DTT], 10% glycerol) and stored at −70°C until use. The 643-bp DNA fragment covering the XylS N-terminal domain (XylS residues 1 to 207) was amplified by PCR from pERD103 plasmid, and the PCR product was digested with the NdeI and XhoI enzymes and subsequently cloned into the pET16b vector (Novagen). The resulting clones were sequenced to confirm XylS-NTD sequence.

Gel filtration chromatography.XylS-CTD molecular weight was estimated by gel filtration chromatography on a Superdex 75 HR 10/30 column (Amersham Biosciences) equilibrated with a mixture of 30 mM Tris-HCl (pH 8), 200 mM KCl, 2 mM DTT, 10% (vol/vol) glycerol at room temperature. A 300-μl solution containing 60 to 100 μM protein was loaded per run. Bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and cytochrome c (12.4 kDa) were used as molecular mass standards. Proteins were detected at 280 nm. Eluted fractions of XylS-CTD were pooled and analyzed by SDS-PAGE (Fig. 2).

• Open in new tab
• Download powerpoint

FIG. 2.

Size exclusion chromatography of the XylS C-terminal domain. The elution profile of XylS-CTD is shown (continuous line). The following molecular mass standards were used: bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and cytochrome c (12.4 kDa). R² = 0.9979. mAU, milli-absorbance units. (Insert) SDS-PAGE of 10 μl of the four 0.5-ml fractions forming the protein peak (numbered as 1, 2, 3, and 4 in the elution profile).

Gel retardation assays (EMSA).Complementary oligonucleotides containing one (AB) or two (ABAB) intact XylS binding sites (Fig. 1) were annealed and blunt-end ligated into HincII-linearized pUC19. DNA probes (130- or 150-bp DNA fragments, respectively) were obtained by PCR amplification of these constructs with M13 universal and reverse primers. The PCR products were isolated from agarose gels using the QIAquick gel extraction kit (Qiagen) and end labeled with [γ-³²P]ATP using T4 polynucleotide kinase (59). Labeled probes (1 nM) were incubated with increasing amounts of purified XylS-CTD domain at 30°C for 15 min in 10 μl of binding buffer [5 mM Tris, 24 mM HEPES, pH 8, 50 mM potassium glutamate, 20 mM NaCl, 1.4 mM EDTA, 0.4 mg/ml bovine serum albumin (BSA), 9% [vol/vol] glycerol, 0.5 μg of poly(dI-dC), and 1 mM DTT]. Samples were loaded onto 8% (wt/vol) nondenaturing polyacrylamide gels in Tris-glycine buffer (0.2 M glycine, 0.025 M Tris-HCl [pH 8.6]). The gels were run for 3 h at 7 V/cm and 4°C, vacuum dried, and exposed to PhosphorImager plates. The results were analyzed using Molecular Imager FX equipment and QuantityOne software (Bio-Rad, Madrid, Spain).

Determination of XylS-CTD DNA stoichiometry by native PAGE.The molecular weight of XylS-CTD-DNA complexes was determined electrophoretically using the procedure of Ferguson (14), as adapted by Orchard and May for protein-DNA complexes (42). Briefly, binding reaction mixtures containing 500 nM to 5 μM XylS-CTD and 50 bp of target DNA or molecular weight markers were analyzed on a series of polyacrylamide gels (7 to 10% [wt/vol] polyacrylamide at 37.5:1) electrophoresed in Tris-glycine buffer (described above) until the bromophenol blue front in flanking sample lanes reached the bottom of the gels. The gels were silver stained. The distance from the loading well to each complex was measured and standardized with the bromophenol blue migration distance to determine the relative mobility (R_f). The logarithm of the R_f was plotted against the gel concentration for each complex and protein standards, and best-fit lines were obtained. The negative slopes of these lines were then plotted against the molecular weights of the protein standards on a double-logarithmic scale, and best-line fit was obtained. The slopes for free DNA and protein-DNA complexes (the CI and CII fast- and slow-migrating complexes, respectively) were depicted, and their molecular weight was extrapolated according to the equation y = 0.1039x + 2.1505. The marker proteins used were carbonic anhydrase, α-lactalbumin, bovine serum albumin dimer, bovine serum albumin monomer, and ovalbumin.

DNase I footprints.A 158-bp DNA fragment covering XylS binding sites (positions from −113 to +35 of the Pm promoter) was specifically radiolabeled at either strand 5′ end using [γ-³²P]ATP and T4 polynucleotide kinase. End-labeled DNA probes (10 nM) were preincubated for 15 min at 30°C in binding buffer with different amounts of purified XylS-CTD. DNase I was added to a final concentration of 0.5 mU/μl and incubated for 5 min at 30°C, and the reaction was stopped by addition of EDTA. Footprint patterns were analyzed on polyacrylamide sequencing gels calibrated with Maxam-Gilbert sequence ladders.

Association constants and cooperativity analysis for XylS-CTD binding.Gel retardation assays of a DNA fragment containing the two XylS binding sites were carried out as described above. The results were quantified using Molecular Imager FX equipment and QuantityOne software (Bio-Rad). The fraction of free DNA (E₀), complex I (E₁), or complex II (E₂) is given by equations 1 to 3. To estimate K_I and K_II, data from gel mobility shift assays were fit to equations 1 to 3 described by Chen et al. (9) using a nonlinear curve-fitting algorithm (Dynamic Curve Fitting from Sigma Plot; Systat Software, Inc.), where P is the total protein concentration and K_I and K_II are the macroscopic association constants for XylS-CTD binding to one or two sites, respectively, of the A1B1A2B2 DNA fragment.

For complex CI, DNA + mP ⇔ DNA-P_m, where K_I = [DNA-P_m]/[DNA][P]^m.

For complex CII, DNA-P_m + nP ⇔ DNA-P_{m + n}, where K_II = [DNA-P_{m + n}]/K_I [DNA][P]^{m + n}. $$mathtex$$\[E_{0}{=}\frac{1}{1{+}K_{\mathrm{I}}[P]^{m}{+}K_{\mathrm{I}}K_{\mathrm{II}}[P]^{m\ {+}\ n}}\]$$mathtex$$(1) $$mathtex$$\[E_{1}{=}\frac{K_{\mathrm{I}}[P]^{m}}{1{+}K_{\mathrm{I}}[P]^{m}{+}K_{\mathrm{I}}K_{\mathrm{II}}[P]^{m\ {+}\ n}}\]$$mathtex$$(2) $$mathtex$$\[E_{2}{=}\frac{K_{\mathrm{I}}K_{\mathrm{II}}[P]^{m\ {+}\ n}}{1{+}K_{\mathrm{I}}[P]^{m}{+}K_{\mathrm{I}}K_{\mathrm{II}}[P]^{m\ {+}\ n}}\]$$mathtex$$(3) where m and n represent the number of protein monomers per site in each case, and equal to 1 in both cases, and DNA-P_m and DNA-P_{m + n} represent CI and CII, respectively.

DNA-bending assays.The pBend2 plasmid derivative pBend2-Pm80 (18) was digested with different enzymes to yield a series of DNA fragments with permuted positions of XylS binding sites. These fragments were end labeled, and gel retardation assays were performed as described above. To estimate the apparent bend angle (α) and to locate the center of the bend, changes in the mobility of the protein-bound DNA fragments (relative to the well position) were fitted to a cosine function (E) (60), (E) cos α/2 = μ_M/μ_E, where μ_M represents the relative migration of the complex with the lowest mobility and μ_E represents the relative migration of the complex with the highest mobility.

β-Galactosidase assays.E. coli CC118λPm::lacZ cells bearing pGP1-2 plasmid were transformed with pET-XylS-C, pET-XylS, pET-XylS-N, or the control plasmid, pET16b (Table 1). Transformants were grown overnight on LB medium containing the appropriate antibiotics. Three independent clones of each strain were used. Quadruplicate cultures were prepared by diluting cells from overnight cultures to 1/100. When cultures had reached an optical density at 600 nm (OD₆₀₀) of 0.4, one of them was maintained as a noninduced control, while the other three were supplemented with 3MB (1 mM), IPTG (0.5 mM), or both, respectively. Samples for β-galactosidase activity determination were taken 1 h after induction. β-Galactosidase activity was determined in permeabilized whole cells according to Miller (38).

XylS-N-terminal domain is responsible of XylS insolubility.As for many members of the AraC family, previous attempts to purify the complete XylS protein were unsuccessful and produced protein aggregates (3, 7, 49, 61). To investigate the basis for this insolubility, we analyzed the two protein domains independently. We found that a His₁₀ fusion protein of the C-terminal domain could be overproduced and directly purified in a soluble form. In contrast, XylS N-terminal domain with a His₁₀ fusion (XylSN) aggregated and formed inclusion bodies under every expression condition tested (12), showing that the NTD was the main contributor to the low solubility of full-length XylS. Since the HTH DNA binding elements of XylS are located at the C-terminal end, we tested if both His-tagged XylS wild-type protein and XylS C-terminal domain were able to activate in vivo expression from the Pm promoter (Table 2). E. coli CC118 (Pm::lacZ, pGP1-2) bears a Pm::lacZ fusion in the chromosome as well as the T7 RNAP gene under the control of the phage λ P_L promoter. This strain was transformed with either pET-XylS or pET-XylS-C, which carry the wild-type or truncated XylS version, respectively, under the control of T7 promoter or with the control plasmid pET16b (Table 1). The values of β-galactosidase activity determined in the presence and absence of 1 mM 3MB showed that in the absence of any inducer, activity was 1,490 ± 132 Miller units (MU) for XylS-CTD protein and 2,290 ± 234 MU in the case of full-length XylS, while in the control strain bearing pET16b the activity was negligible (Table 2). These high effector-independent levels correspond to high basal T7 polymerase-dependent XylS expression. It is well established that the high level of XylS protein obtained from a high-copy-number vector induces Pm transcription in the absence of effector (45, 49). Activity in the E. coli CC118 (Pm::lacZ, pGP1-2) strain transformed with pET-XylSN was negligible under all conditions (6 MU) and similar to values obtained with the control plasmid, pET16b. In E. coli strain CC118 (Pm::lacZ, pGP1-2), carrying the XylS-CTD fusion protein gene, no significant increase in Pm activity was detected in the presence of either 3MB or IPTG, while in the case of full-length XylS, the addition of 3MB promoted a 2-fold increase in Pm expression level (Table 2). These results confirm that both the DNA-binding domain devoid of the N-terminal domain and the full-length XylS are capable, when overexpressed, to activate transcription even in the absence of effector (19, 28). However, our results demonstrate that the 3MB response relies solely on XylS N terminal.

Pm half-sites are necessary and sufficient for XylS binding.Most AraC family members appear to form dimers in solution, and dimerization determinants have been proposed to reside in the N-terminal domain (6, 8, 43, 44, 49). If this were the case for XylS, a truncated protein devoid of its N-terminal domain would behave as a monomer in solution. We determined the XylS-CTD oligomeric state using gel filtration through a Superdex G-75 column as described in Materials and Methods. A single peak was detected at an elution volume corresponding to a molecular mass of 16 kDa, in good correlation with the molecular mass of XylS-CTD (16.81 kDa), thus implying that the protein was a monomer under our experimental conditions (Fig. 2).

We next examined XylS-CTD binding to its operator, defined as two binding sites organized as direct repeats, one overlapping the RNA polymerase binding site (proximal site) and the second one located 6 nucleotides upstream (distal site). Each binding site is composed of two different boxes, A1/A2 and B1/B2, conserved in both sites and separated by a sequence of six nonconserved nucleotides (13, 19) (Fig. 1). To test if a single binding site was sufficient for XylS-CTD binding, a DNA fragment containing the distal XylS binding site composed of A1 and B1 half-sites was titrated with increasing XylS-CTD concentrations. A single shifted band was observed, corresponding to complex CI (Fig. 3a). As expected (12, 28), when an A1B1A2B2 DNA fragment containing the complete two-site wild-type binding region was titrated, a second, slower-migrating shifted band was formed (complex CII) (Fig. 3b). The figure shows that as the XylS-CTD concentration increased, the CI complex was progressively replaced by the lower-mobility CII complex.

• Open in new tab
• Download powerpoint

FIG. 3.

XylS-CTD binding to Pm operator. (a) Electrophoretic mobility shift assay of XylS-CTD binding to a Pm template containing only one binding site, symbolized as A1B1 (Fig. 1). The labeled DNA fragment (1 nM) was incubated with 50 nM, 100 nM, 200 nM, 400 nM, 600 nM, 1 μM, 2 μM, 4 μM, and 8 μM purified XylS-CTD (lanes 2 to 10, respectively). Lane 1, free DNA. One XylS-CTD-DNA complex was formed (CI). The scheme depicts the DNA fragment used in the assay, including only one binding site (arrow) composed of A1 (black) and B1 (gray) half-sites. (b) Electrophoretic mobility shift assay of XylS-CTD binding to A1B1A2B2 template. The labeled DNA fragment was incubated with 50 nM, 100 nM, 200 nM, 400 nM, 600 nM, 1 μM, 2 μM, 4 μM, and 8 μM purified XylS-CTD (lanes 2 to 10, respectively). Lane 1, free DNA. XylS-CTD-DNA complexes CI and CII were formed. The scheme depicts DNA fragment used in the assay, including distal (D) and proximal (P) binding sites (arrows), each composed of A (black) and B (gray) boxes. A small arrow indicates the loading wells in the gel.

One XylS-CTD monomer per single binding site.To determine the DNA/protein stoichiometry of CI and CII complexes, we measured the molecular weight of the two complexes using the electrophoretic method adapted by Orchard and May (42), based on changes in gel mobility at different acrylamide concentrations. Briefly, gel mobility of CI and CII complexes and of a series of molecular weight markers was first plotted against polyacrylamide concentration, and slopes were calculated in each case (Fig. 4a). The linear regression slopes obtained for each molecular weight marker were plotted against the logarithm of the molecular weight (Fig. 4b). When slope values of protein-DNA complexes CI and CII were fitted to the plot, an apparent molecular mass of 46 kDa was estimated for the CI XylS-CTD-DNA complex. The molecular mass of the protein component of the CI complex was extrapolated as 17 kDa after subtracting 29 kDa corresponding to the apparent molecular mass of the 50-bp A1B1A2B2 DNA fragment, consistent with CI resulting from a single XylS-CTD monomer (16.81 kDa) bound to the promoter probe. Likewise, an apparent molecular mass of 57 kDa was obtained for CII complex (28 kDa corresponding to the protein component), showing a slight deviation from the expected mass of 33.62 kDa for two XylS-CTD monomers.

• Open in new tab
• Download powerpoint

FIG. 4.

Determination of the stoichiometry of XylS-CTD-DNA complexes by native PAGE. Protein markers and XylS bound to DNA sequence were electrophoresed at different polyacrylamide concentrations. (a) The logarithms of the relative mobilities of XylS-CTD-DNA complexes and marker proteins with respect to bromophenol blue were first plotted as a function of polyacrylamide concentration. (b) The slope of the regression equation obtained for each marker is plotted against their molecular weight (MW). The values for free DNA (black squares), CI (light gray triangles), and CII (dark gray diamonds) are depicted, and their molecular weights were extrapolated according to the equation y = 0.1039x + 2.1505. The marker proteins used were carbonic (c.) anhydrase, α-lactalbumin, bovine serum albumin dimer, bovine serum albumin monomer, and ovalbumin.

Binding of XylS-CTD to the Pm promoter induces bending.Previous work showed that Pm promoter DNA region was intrinsically curved, with an apparent bent angle of 35° ± 5° (16, 18, 19). To explore if binding of XylS-CTD induced further distortion in the DNA, we applied the circular permutation assay developed by Kim and coworkers (30, 64) (see Materials and Methods). A series of 195-bp DNA fragments with permuted positions of the XylS binding sites were incubated with XylS-CTD, and the mobility of the two complexes (CI and CII) was analyzed by native polyacrylamide gel electrophoresis (64) (Fig. 5). The intrinsic bend angles (α) and the location of the center of the bend were estimated according to Thompson and Landy (60). Although this method does not resolve the degree of curvature at each single position in the DNA, we could observe that the Pm intrinsic curvature centered within the A track spanning positions −41 to −46 increased to a global value of 50° ± 5° after binding of one XylS-CTD monomer and to 98° ± 2° after binding of two monomers (Fig. 5). The new center of the bent region was located between positions −49 and −52 in both CI and CII complexes, evidencing a shift from the previously determined free DNA bending center within the XylS binding site toward the DNA region between the two binding sites (Fig. 1 and 5).

• Open in new tab
• Download powerpoint

FIG. 5.

XylS-CTD binding to DNA provokes DNA bending. Gel retardation assays of XylS-CTD on a series of 195 bp DNA fragments, containing XylS binding sites in permutated locations were run in 8% polyacrylamide (wt/vol). The migration distance of each complex (*, CI; ▴, CII) was plotted against the distance between XylS binding site 5′ end and fragment 5′ end in each fragment tested. The bending angle for each complex was estimated according to the equation cos α/2 = μ_M/μ_E, where μ_M is the migration of the complex with lowest mobility and μ_E is the migration of the complex with highest mobility. Values are the average of six independent determinations.

Stepwise binding of XylS-CTD to Pm binding sites.In mobility shift assays, we observed that at a low XylS-CTD concentration, CI was preferentially formed, followed by a progressive accumulation of CII concomitant with a decrease of CI at a higher protein concentration (Fig. 3). To analyze if this could result from the first monomer at the proximal site facilitating binding of the second one in a cooperative manner, we performed a series of quantitative analyses of the binding interactions. To this end, five repetitions of titration assays of a DNA fragment containing one or two binding sites with XylS-CTD were performed and the amounts of CI and CII complexes of XylS-CTD bound to an A1B1A2B2 fragment were plotted as a function of XylS-CTD concentration (Fig. 6; see equations 1 to 3 in Materials and Methods) (9). XylS binding sites are not identical in sequence; thus, separate estimates of intrinsic binding affinity for each site could not be derived from two-site gel shift experiments. We used equations 1 to 3 as derived by Chen et al. (9) to estimate the macroscopic association constants, K_I and K_II. The average data were fit to equations 1 to 3, and K_I and K_II constants were derived using nonlinear curve fitting (see Materials and Methods). The data obtained (K_I = 1.56 × 10⁶ M⁻¹ and K_II = 7.3 × 10⁵ M⁻¹) are not supportive of significant cooperative interaction in the sequential binding of two XylS-CTD monomers to their recognition sites.

• Open in new tab
• Download powerpoint

FIG. 6.

Analysis of XylS-CTD binding to Pm. (a) Representative EMSA of XylS-CTD binding to the DNA containing the XylS binding sites shown under the gel as a scheme. The XylS-CTD concentrations were 0, 80 nM, 400 nM, 1.5 μM, 2.5 μM, and 5 μM. CI, complex 1; CII, complex 2; F, free DNA. A small arrow indicates the loading wells in the gel. (b) Quantitation average of gel shift assay data from five independent experiments. The free DNA (black circles), CI complex (blue), and the CII complex (red) were quantified in a PhosphorImager. Lines represent the best fit of the data to equations 1 to 3 (equation 1, black; equation 2, blue; and equation 3, red [see Materials and Methods]). The regression coefficients for each fit were R_free = 0.952, R_CI = 0.957, and R_CII = 0.978. Error bars correspond to standard deviation values.

To analyze if XylS-CTD monomer preferentially bound any of its binding sites, we carried out DNase I footprinting assays of XylS-CTD bound to a Pm fragment. Figure 7 shows that XylS-CTD protected two regions centered at positions −60 and −40, corresponding to the two 15-bp direct repeats (distal and proximal sites). We observed that at a low protein concentration, the proximal site was fully protected, whereas the distal site was only progressively protected by increasing XylS-CTD concentration (see especially the bottom strand footprinting in Fig. 7b). Interestingly, a hyperreactive band corresponding to a T residue at position −42, visible in free DNA, almost disappeared at low XylS-CTD concentrations (Fig. 7a). Concomitant with this, a band corresponding to a T residue at position −51 became hyperreactive, suggesting a shift in the bending center at the Pm promoter (Fig. 7a). These results support sequential and directional binding of the two XylS-CTD monomers at Pm, generating a progressive curvature in the DNA between the two binding sites. Surprisingly, the hyperreactive band had not been previously observed except as a very weak signal in footprint assays using an antigen-tagged XylS C-terminal domain derivative (28).

• Open in new tab
• Download powerpoint

FIG. 7.

DNase I footprint analysis of XylS-Pm complexes. (a) Upper strand; (b) bottom strand. Lane 1, free DNA. The XylS-CTD concentrations used were as follows: (a) 1 μM (lane 2), 2 μM (lane 3), 5 μM (lane 4), and 10 μM (lane 5); (b) 500 nM (lane 2), 1 μM (lane 3), and 2 μM (lane 4). The position of the region corresponding to the distal (D) and proximal (P) sites is indicated. BC, bending center. The band position in free DNA was determined by comparison with a Maxam and Gilbert sequence ladder (not shown).