The Phusion High-Fidelity DNA polymerase (Thermo Scientific) was used. Reaction mixtures (50 μl) contained 5–20 ng of template DNA, 20 pmol of each primer, 200 μM each deoxynucleoside triphosphate (dNTP), and one unit of DNA polymerase. A initial denaturation step was performed at 98°C for 1 min, followed by 30 cycles that included the next steps: (i) denaturation at 98°C for 10 s, (ii) annealing of the primers around 55°C (depending on the primer melting temperature) for 20–30 s, and (iii) extension at 72°C for 20–40 s (depending on the amplicon length). A final extension step was performed at 72°C for 10 min. PCR products were cleaned up with the QIAquick PCR purification kit (Qiagen).

Oligonucleotides used for PCR amplifications are listed in Table 1. As DNA templates, chromosomal DNA from the pneumococcal R6 strain (Hoskins et al., 2001) and pANN202312R plasmid DNA from E. coli (Godessart et al., 1988) were used. Chromosomal DNA from S. pneumoniae was prepared as described previously (Ruiz-Cruz et al., 2010). For small-scale preparations of plasmid DNA, the High Pure Plasmid Isolation Kit (Roche Applied Science) was used. The 222-bp DNA region of the R6 chromosome (coordinates 1598298–1598519) was amplified using the 1622H and 1622I primers. The 288-bp hlyR DNA fragment (coordinates 129–416 in Madrid et al., 2002) was amplified using the hlyR-Fw and hlyR-Rev primers. The 290-bp hlyC DNA fragment (coordinates 2109–2398 in Madrid et al., 2002) was amplified using the hlyC-Fw and hlyC-Rev primers.

Oligonucleotides were radioactively labeled at the 5′-end using [γ-³²P]ATP (PerkinElmer) and T4 polynucleotide kinase (T4 PNK; New England Biolabs). Reactions (25 μl) contained 30 pmol of oligonucleotide, 2.5 μl of 10 × kinase buffer (provided by the supplier), 50 pmol of [γ-³²P]ATP (3000 Ci/mmol, 10 mCi/ml) and 10 units of T4 PNK. After incubation at 37°C for 30 min, additional T4 PNK (10 units) was added. Reaction mixtures were then incubated at 37°C for 30 min. The enzyme was inactivated by incubation at 65°C for 20 min. Non-incorporated nucleotide was removed using Illustra MicroSpin™ G-25 columns (GE Healthcare). The 5′-labeled oligonucleotides were used for manual sequencing and for PCR amplification to obtain double-stranded DNA fragments labeled at either the coding or the non-coding strand.

Gene mgaSpn was engineered to encode a His-tagged MgaSpn protein (MgaSpn-His). This variant of MgaSpn carries six additional His residues at the C-terminal end. The procedure used to overproduce and purify MgaSpn-His was reported previously (Solano-Collado et al., 2012). Purified H-NS-His protein was obtained as described by Nieto et al. (1991).

Binding reactions (10 μl) contained 40 mM Tris-HCl, pH 7.6, 1 mM DTT, 0.4 mM EDTA, 1–2% glycerol, 50 mM NaCl, 10 mM MgCl₂, 500 μg/ml bovine serum albumin (BSA), 2 nM of 5′-labeled DNA and varying concentrations of MgaSpn-His (20 to 180 nM). Reactions were incubated at ambient temperature for 20 min. Free and bound DNA forms were separated on native polyacrylamide (5%) gels (Mini-PROTEAN system, Bio-Rad) using Tris-borate-EDTA, pH 8.3, buffer (TBE). Gels were run at 100 V and ambient temperature. Labeled DNA was visualized using a Fujifilm Image Analyzer FLA-3000 and quantified using the Quantity One software (Bio-Rad).

For competitive EMSA, a 407-bp DNA fragment from the 5′ untranslated region of the hlyR gene was generated by PCR using primers Hly Sal/Eco5 and Hly Sal/Hind3 (Table 1). For each reaction, 50 ng of the pneumococcal 222-bp DNA and 150 ng of the 407-bp DNA (competitor DNA) were mixed with increasing concentrations of H-NS-His in binding buffer (250 mM HEPES, pH 7.4, 350 mM KCl, 5 mM EDTA, 5 mM DTT, 500 μg/ml BSA, 25% glycerol) and incubated at 37°C for 30 min. Samples (20 μl) were loaded onto native polyacrylamide (5%) gels (TBE buffer). Bands were visualized using a Gel-doc system (Bio-Rad).

In the case of H-NS-His, binding reactions (10 μl) contained 30 mM Tris-HCl, pH 7.6, 1 mM DTT, 1 mM CaCl₂, 10 mM MgCl₂, 100 mM NaCl, 1% glycerol, 2 nM ³²P-labeled DNA and different concentrations of H-NS-His (2 nM to 500 nM). For MgaSpn-His, binding reactions (10 μl) contained 40 mM Tris-HCl, pH 7.6, 1.2 mM DTT, 0.2 mM EDTA, 1 mM CaCl₂, 10 mM MgCl₂, 50 mM NaCl, 1% glycerol, 500 μg/ml BSA, 2 nM ³²P-labeled DNA and different concentrations of MgaSpn-His (10 to 200 nM). In all cases, after 20 min at ambient temperature, 0.03 units of DNase I (Roche Applied Science) was added and the reaction proceeded for 5 min at the same temperature. DNase I digestion was stopped by adding 1 μl of 250 mM EDTA. Then, 4 μl of loading buffer (80% formamide, 1 mM EDTA, 10 mM NaOH, 0.1% bromophenol blue and 0.1% xylene cyanol) was added. After heating at 95°C for 5 min, samples were loaded onto 8 M urea-6% polyacrylamide gels. Dideoxy-mediated chain termination sequencing reactions were run in the same gel. Labeled products were visualized using a Fujifilm Image Analyser FLA-3000. The intensity of the bands was quantified using the Quantity One software (Bio-Rad).

The bendability/curvature propensity plots of the DNA fragments used in this study were calculated with the bend.it server (Vlahovicek et al., 2003; http://hydra.icgeb.trieste.it/dna/bend_it.html) as described previously (Solano-Collado et al., 2013).

In the E. coli plasmid pHly152, previous DNase I footprinting assays revealed the existence of two extended H-NS binding sites upstream of the hly operon (sites I and II in Figure 1A) (Madrid et al., 2002). One of them (site I; coordinates 190–350 of pHly152) is included within the so-called hlyR regulatory sequence (Vogel et al., 1988), and was shown to play a significant role in the thermoregulation of the hly operon (Madrid et al., 2002). To investigate whether the pneumococcal MgaSpn protein is able to recognize particular regions on the H-NS binding site I, we performed EMSA and DNase I footprinting experiments. We used a His-tagged version of MgaSpn (MgaSpn-His) and a 288-bp DNA fragment (here named hlyR; coordinates 129–416) that contains the site I (Figure 1A). The presence of a His-tag at the C-terminal end of MgaSpn does not affect its DNA-binding properties (Solano-Collado et al., 2012, 2013). For EMSA, radioactively labeled DNA was incubated with increasing concentrations of MgaSpn-His (Figure 2A). At 20 nM of MgaSpn-His, free DNA and two protein-DNA complexes were detected. However, in agreement with previous results (Solano-Collado et al., 2013), as the concentration of MgaSpn-His was increased, complexes of lower electrophoretic mobility appeared sequentially and complexes moving faster disappeared gradually, indicating that multiple protein units bind orderly on the same DNA molecule (formation of multimeric protein-DNA complexes).

By EMSA, we estimated the affinity of MgaSpn-His for the 288-bp hlyR DNA fragment (Figure 2B). Since MgaSpn-His generates multiple protein-DNA complexes, the protein concentration required to bind half the DNA was determined by measuring the decrease in free DNA rather than the increase in complexes, which gives an indication of the approximate magnitude of the dissociation constant, Kd (Carey, 1988). Such a concentration was about 50 nM. This value is similar to the apparent Kd of MgaSpn for the pneumococcal 222-bp DNA fragment that harbors the PB activation region (MgaSpn binding site) (see Figure 1B) (Solano-Collado et al., 2013).

The position of MgaSpn-His on the hlyR DNA fragment was further analyzed by DNase I footprinting assays (Figure 3). The DNA fragment was labeled either at the 5′-end of the coding strand or at the 5′-end of the non-coding strand. On the coding strand and at 40 nM of MgaSpn-His, protections against DNase I digestion were observed at a particular region (from position 242 to 263) (Figure 3A). Diminished cleavages were also observed from position 342 onwards (no resolution in the gel). Moreover, positions 227, 280, and 341 were more sensitive to DNase I cleavage. On the non-coding strand and at 20 nM of MgaSpn-His, diminished cleavages were observed from 243 to 266, from 345 to 354, and from 366 to 379 (Figure 3A). Additionally, the 278 and 381 positions were more sensitive to DNase I cleavage. These results indicated that MgaSpn-His recognizes preferentially two sites on the hlyR DNA fragment (Figure 3B). One of them (site A; positions 242 to 266) is located within the H-NS binding site I, whereas the other one (site B, positions 342 to 379) is adjacent to it. On both strands and at 80 nM of MgaSpn-His, regions protected against DNase I digestion were observed along the DNA fragment (Figure 3A), which is consistent with the pattern of protein-DNA complexes observed by EMSA (Figure 2A).

Figure 4A shows the bendability/curvature propensity plot of the 288-bp hlyR DNA fragment (coordinates 129–416) according to the bend.it program (Vlahovicek et al., 2003). The profile contains a peak of potential sequence-dependent curvature at position 238, just adjacent to the MgaSpn-His binding site A (positions 242-266). Its magnitude (9.7 degrees per helical turn) is within the values calculated for experimentally tested curved motifs (Gabrielian et al., 1997). Furthermore, the profile reveals that site A is flanked by regions of potential bendability (positions 215-222 and 271-278). The MgaSpn-His binding site B (positions 342-379) contains a peak of predicted curvature at position 364 (magnitude 14) which is also flanked by regions of potential bendability (positions 319-335 and 380-396). Thus, on the hlyR DNA fragment (see Figure 1A), MgaSpn-His binds preferentially to two sites (sites A and B) that are flanked by regions of potential bendability. Whereas, site A is located within the extended H-NS binding site I, site B is just adjacent to it.

H-NS interacts not only with the site I of plasmid pHly152 but also with the site II (coordinates 2180–2330), which is located upstream of hlyC, the first gene of the hly operon (Madrid et al., 2002) (see Figure 1A). Site II includes two of the three promoters described for the hly operon (Koronakis and Hughes, 1988). To analyze whether MgaSpn-His recognizes particular regions on the H-NS binding site II, we used a 290-bp DNA fragment (here named hlyC; coordinates 2109–2398) that contains the site II at internal position (Figure 1A). Radioactively labeled DNA was incubated with increasing concentrations of MgaSpn-His. By EMSA, we found that MgaSpn-His also generates multimeric complexes on the hlyC DNA fragment (Supplementary Figure 1). The apparent Kd of MgaSpn-His for the hlyC DNA fragment was about 75 nM, slightly higher than for the hlyR DNA fragment (Figure 2B).

The position of MgaSpn-His on the hlyC DNA fragment was further analyzed by DNase I footprinting assays (Figure 5A). To this end, the 290-bp DNA fragment was labeled at the 5′-end of the coding strand. At 60 and 80 nM of MgaSpn-His, diminished DNase I cleavages were mainly observed from coordinate 2197 to 2231 (site C), from 2251 to 2269 (site D), and from 2293 to 2304 (site E). Moreover, positions 2188, 2235, 2325, and 2336 were more sensitive to DNase I digestion. At higher protein/DNA ratios, MgaSpn-mediated protections were observed along the hlyC DNA fragment (Figure 5A). Therefore, MgaSpn-His recognizes preferentially three regions within the extended H-NS binding site II (Figure 5B). Compared to the 288-bp hlyR DNA fragment (Figure 4A), the magnitude of the curvatures predicted in the 290-bp hlyC DNA fragment is slightly lower (<9 degrees per helical turn) (Figure 4B). Nevertheless, the MgaSpn-His binding sites D (2251-2269) and E (2293-2304) are located at regions of conspicuous bendability (positions 2248-2267 and 2296-2309, respectively).

In vivo activation of the P1623B promoter requires a 70-bp region (PB activation region) located upstream of the promoter (from position −50 to −119) (Figure 1B). By DNase I footprinting experiments, we demonstrated previously that MgaSpn-His interacts with the PB activation region (positions −52 to −102) when it is located at internal position on a 222-bp DNA fragment (coordinates 1598298 to 1598519 of the pneumococcal R6 genome; see Figure 1B) (Solano-Collado et al., 2012). Similar results were obtained using an untagged form of the MgaSpn protein (Solano-Collado et al., 2013). In the present study, we analyzed whether protein H-NS-His is able to bind to the pneumococcal 222-bp DNA fragment that contains the PB activation region. First, we performed a competitive gel retardation assay (Figure 6). The pneumococcal 222-bp DNA fragment was mixed with a 407-bp DNA fragment (competitor DNA) from the E. coli plasmid pHly152, and both DNAs were incubated with increasing concentrations of H-NS-His. The 407-bp DNA fragment was reported to lack preferential binding sites for H-NS (Madrid et al., 2002). As shown in Figure 6, protein H-NS-His showed a higher affinity for the pneumococcal 222-bp DNA fragment. Next, we used DNase I footprinting to identify the sites recognized by H-NS-His. The pneumococcal 222-bp DNA fragment was radioactively labeled at the 5′-end of the non-coding strand, and then it was incubated with increasing concentrations of H-NS-His (Figure 7A). At 10 nM of H-NS-His, changes in DNase I sensitivity (diminished cleavages) were observed from positions −53 to −68 (site 1), −102 to −111 (site 2), and −121 to −131 (site 3). Whereas, site 1 and site 2 are located within the PB activation region, site 3 is adjacent to it (Figure 7B). At 80 nM of protein, H-NS-His mediated protections were observed along the entire DNA fragment (Figure 7A). According to predictions of intrinsic DNA curvature (Solano-Collado et al., 2013), the pneumococcal 222-bp DNA fragment contains one peak of potential curvature (magnitude 9.5, position −90) within the PB activation region (Figure 1B). Furthermore, there are two regions of potential bendability (from −62 to −76 and from −110 to −122) flanking such a curvature. Hence, the three sites recognized by H-NS-His on the pneumococcal 222-bp DNA fragment are adjacent to regions of potential bendability.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.