MalR-mediated Regulation of the Streptococcus pneumoniae malMP Operon at Promoter PM

INFLUENCE OF A PROXIMAL DIVERGENT PROMOTER REGION AND COMPETITION BETWEEN MalR AND RNA POLYMERASE PROTEINS*

Concepción NietoDagger , Antonio Puyet§, and Manuel EspinosaDagger

From the Dagger  Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, E-28006 Madrid and the § Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, E-28040 Madrid, Spain

Received for publication, December 4, 2001, and in revised form, February 7, 2001

    ABSTRACT
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The Streptococcus pneumoniae mal regulon contains two operons, malXCD and malMP involved in the uptake and utilization of maltosaccharides. Both operons are transcribed from two divergent promoters, PX and PM, and are negatively regulated by the MalR transcriptional repressor. Purified MalR protein binds to two DNA regions that encompasses both promoters, thus occupying its two operators, OM and OX. However, the levels of occupation and repression were different, being higher when MalR was bound to OM than when it was anchored to OX. Competition experiments between MalR and the Escherichia coli RNA polymerase on promoters PM and PX showed that the affinity of either protein for the promoter/operator DNA sequences was important to determine the frequency of transcription initiation. In addition to the control exerted by MalR, expression from promoter PM was affected by upstream sequences located within or close to PX promoter.

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Initiation of transcription in prokaryotes is the stage usually controlled by positive and negative regulators (1-3). In many of the known instances of repressor proteins, they generally bind to specific DNA sequences, which are located close to or within the promoter, although the binding position of the repressors seems to vary, in contrast to the relatively fixed binding positions of the activator sites (4). As a consequence, repressors may hinder the binding of the RNA polymerase (RNAP)1 to the promoter, thus interfering with the transcription initiation process (3, 5). The mechanisms of transcription inhibition by repressor proteins have been mainly studied in the case of Gram-negative bacteria, especially in Escherichia coli (6) and in some of its extrachromosomal genetic elements, in which examples of inhibition of transcription at different stages have been reported. Repression by phages P22-Arc and lambda l-cI proteins is exerted at the first step of initiation. These proteins compete with RNAP for binding to the free promoter, although mutational analyses have shown that both proteins may exhibit a more complex behavior (7, 8). A similar picture has been reported for the lac repressor, in which the binding of the protein to its DNA target hinders the access of RNAP to the promoter (9), whereas in the case of plasmid R6K-encoded KorB repressor, inhibition occurs at the isomerization stage (10). Repression mediated by GalR is due to a GalR-induced DNA loop at the promoter region, the protein inhibiting synthesis of both abortive products, and complete transcripts (11). There are more complex situations, like the AraC regulatory protein, in which the three AraC-DNA binding sites are positioned so that the protein can repress or activate the promoters located in the araCBAD region via DNA looping (see Ref. 12, and references therein). In the case of Gram-positive bacteria, several chromosome-encoded transcriptional repressors have been described, mainly from Bacillus subtilis (13-15), Staphylococcus aureus (16-18), and Streptomyces coelicolor (19). However, information on their mechanism of repression at the molecular level is still scarce.

Over a number of years, we have been studying the mal regulon of the Gram-positive bacterium Streptococcus pneumoniae (Fig. 1). This regulon is composed of three operons, two of them involved in maltosaccharide uptake (malXCD) and its utilization (malMP), the third one (malAR) being involved in regulation of the other two operons (20, 21). The two former operons are transcribed from two divergently oriented promoters, termed PM (for the malMP operon) and PX (for the malXCD operon), which are negatively regulated by the product of gene malR (22). Protein MalR belongs to the LacI-GalR family of transcriptional repressors (23) and binds specifically to two operator sequences located in the intergenic region between operons malXCD and malMP (22). However, purified MalR protein was shown to bind more tightly to the malMP operator sequence (OM) than to the malXCD (OX), even though both operators differ only by two nucleotides. The binding of MalR to its DNA target is inactivated by the addition of maltose (22).

In the present work, we have studied the occupancy of the promoter/operator regions of the malMP and malXCD operons by purified MalR and RNAP proteins through in vitro transcription, electrophoretic mobility shift assays (EMSA), and DNase I protection. Identification of the initiation of transcription sites for both operons showed that promoters PM and PX are in the vicinity of two palindromic DNA sequences (the OM and OX operators, respectively), which are the sites where MalR protein binds (22). The target sites of MalR and RNAP overlapped, and both proteins competed for their binding to DNA. Affinities of MalR and RNAP for binding to their respective DNA sequences were important for the level of gene expression of both operons. In addition, gene fusions showed that neither promoter PX nor operator OX were needed for in vivo expression of the malMP operon, and that this region was also unnecessary for MalR-mediated repression of PM. However, the DNA region upstream of the malMP promoter/operator region may play a role in modulation of transcription from PM, as shown by mutational analyses of the PX/OX DNA region.

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Bacterial Strains and Plasmids-- S. pneumoniae R61 (wild type) harboring plasmid pLS70 was used for preparation of total RNA. Overexpression of the malR gene was performed in E. coli BL21(DE3) using the pET21-b (Novagen)-derived plasmid pMRWT, in which the malR gene is cloned as a fusion protein harboring a C-terminal His6 tag. Plasmid pLS70 contains a 3.5-kilobase pair PstI DNA fragment of the S. pneumoniae chromosome cloned into the streptococcal plasmid pMV158 and harbors part of the mal regulon including promoters PX and PM (24). Plasmid pLS1MGFP was constructed like the previously described pLS1GFP (25), but digesting the parental pCL1GFP with SalI and HindIII, cloning this fragment into plasmid pJDC9 (26), and then into pLS1 (27). In plasmid pLS1MGFP, the sequence encompassing promoter PX is removed, and the reporter gfp gene (encoding the green fluorescent protein (GFP)) is placed under the control of promoter PM. The nucleotide sequences of the pneumococcal inserts in these plasmids, pLS1GFP and pLS1MGFP, were determined and shown to be identical to the corresponding pneumococcal chromosomal sequence. In addition to the above, plasmid pLS1Er was constructed by substitution of the EcoRI-HindIII fragment of pLS1 by a ClaI-SmaI fragment of pJDC9, so that the cloned fragment harbors the erm gene (encoding resistance to erythromycin (Er)). Selection was applied for resistance to tetracycline (1 µg/ml; pLS70, pLS1GFP, pLS1MGFP, and pLS1mXGFP), erythromycin (1 µg/ml; pLS1Er, pLS1GFP, pLS1MGFP, and pLS1mXGFP), or ampicillin (200 µg/ml; pMRWT).

Oligonucleotides and Polymerase Chain Reaction Amplification-- The following oligonucleotides were used in the PCR amplification reactions to obtain DNA templates, either for EMSA assays (oligonucleotides 1-4) or for site-directed mutagenesis (mut1, mut2, mal3, and mal4). Their co-ordinates (22) are given in parentheses: 1, 5'-GTGTAACAGTTCCAAGCACCG-3' (1170-1190); 2, 5'-TCCGATTCCGTAAGCTCCTGG-3' (1832-1812); 3, 5'-GGGATTAGAACCAGGGAGGTA-3' (1487-1467); 4, 5'-TACCTCCCTGGTTCTAATCCC-3' (1467-1487); mut1, 5'-GCAACCGTTTTCTATTTGCACCCTACTAAGCTCATAAAG-3' (1313-1275); mut2, 5'-CTTTATGAGCTTAGTAGGGTGCAAATAGAAAACGGTTGC-3' (1275-1313); mal3, 5'-GCAGAATTCAAGTTTTATTGATAAGGAAAC-3 (1241-1262); mal4, 5'-CGCGGATCCATCTCTAGAGTATTTTGCAGACGCAAACG-3' (1730-1711).

The latter two oligonucleotides contained recognition sites (underlined) for the restriction enzymes EcoRI (mal3), and BamHI and XbaI (mal4).

The three DNA fragments used, namely XM, X, and M, were amplified by PCR using oligonucleotides 1-2, 1-3, or 2-4, respectively. Amplification was done during 20 cycles using the Pfu DNA polymerase (Statagene) and, as template, plasmid pLS70 DNA (22). The fragments obtained had blunt ends. When the fragments were used for DNase I footprinting assays, synthesis of the DNA fragments by PCR was carried out after 5'-end labeling of one of the primers with [gamma -32P]ATP and polynucleotide kinase (28).

Overproduction and Purification of MalR-- To increase the solubility of the MalR protein, we modified the method previously described (22). To this end, E. coli BL21(DE3) cells harboring plasmid pMRWT were induced as described (21). Cells were suspended in buffer A containing 20 mM Na2HPO4, pH 7.6, supplemented with 1 M NaCl, and disrupted by passage through a French pressure cell. The cell lysate was cleared by low and high speed centrifugation, and the supernatant was passed through an immobilized metal ion affinity chromatography column (chelating sepharose fast flow, Amersham Pharmacia Biotech), eluting the protein with buffer B (1 M NaCl, 20 mM Na2HPO4, pH 7.6) containing 500 mM imidazole. The eluate, containing MalR purified almost to homogeneity, was dialyzed against buffer C (250 mM NaCl, 20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% ethylene glycol), and stored at -80° °C. No loss of DNA binding activity was observed during 1-year storage.

Mapping of Initiation of Transcription Start Points and in Vitro Transcription Assays-- Total RNA was isolated from S. pneumoniae R61, and endonuclease S1 protection and primer extension assays were performed as described (23). The in vitro transcription assays were carried out as templates the three DNA fragments (X, M, or XM), which harbor promoters PX, PM, or both, respectively. Transcription reactions (50 µl) contained 2 nM amounts of template DNA, ATP, CTP, GTP (200 µM each), UTP (55 µM), and 0.25 µM [alpha - 32P]UTP (2.5 µCi), 12 units of ribonuclease inhibitor (RNasin, Roche Molecular Biochemicals), 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 200 mM NaCl, 0.1 mM EDTA, 5% glycerol, and 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech). Reactions were started by the addition of RNAP (purchased from Roche Molecular Biochemicals) (0.165 units; 22 nM). When the effect of MalR on transcription was assayed, the mixtures received this protein at the concentrations indicated under "Results and Discussion." When RNAP was added before MalR, the NTPs and the poly(dI-dC) were omitted in the reaction mixtures, and they were added simultaneously to the MalR protein. Reaction mixtures were incubated 15 min at 37° °C, and then stop buffer (final concentration of 300 mM sodium acetate, pH 8.0, 15 mM EDTA, and 0.1 µg/µl of tRNA) was added. Nucleic acids were ethanol-precipitated, and the transcripts were separated by 6% PAGE, 8 M urea sequencing gels. Results were quantified from three different experiments by means of the storage phosphor technology, with the aid of a PhosphorImager equipment and the ImageQuant software (Molecular Dynamics). The approximate size of the run-off transcripts were determined by comparison to the length of the sequence ladder of the same DNA template.

DNase I Footprint Experiments-- The XM-DNA fragment (synthesized by PCR) was 5'-end labeled. Reactions were done in 50 µl of buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 2.75 mM CaCl2, 1 mM dithiothreitol, 0.1 mM EDTA), supplemented with 200 mM NaCl and 500 ng of calf thymus DNA. Mixtures were incubated with RNAP (Roche Molecular Biochemicals, at the specific activity of 1 unit/µl; 200-400 units/mg of protein) and/or MalR proteins (37 °C, 15 min), prior to digestion with 0.042 units of DNase I (Worthington, 2.15 units/µl), 5 min at room temperature. Reactions were stopped by addition of 25 µl of stop buffer (2 M ammonium acetate pH 7.5, 0.15 M EDTA, 0.8 M sodium acetate, pH 7, 0.1 µg/µl calf thymus DNA, and 0.4 µg/µl tRNA). Samples were ethanol-precipitated, and the DNA products were separated by 6% denaturing polyacrylamide gel electrophoresis (28).

EMSA with MalR and/or RNAP Proteins-- DNA binding reactions (30 µl) contained 20 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 10 mM MgCl2, 200 mM NaCl, and 5% glycerol. Purified MalR protein (0.225-0.900 µM) and/or RNAP (36 nM) was mixed with 32P-labeled DNA (0.9 nM), and 1 µg of poly(dI-dC). Reaction mixtures were incubated at 37° °C, 15 min. Free and bound DNA forms were separated by native PAGE, using 5% gels for the assays with the X (318-bp) or the M (365-bp) DNA fragments, and 4% gels when the XM (664-bp) DNA fragment was used. To measure the half-lives of RNAP-DNA, 36 nM RNAP were added to a solution containing 0.9 nM 32P-labeled X or M DNA fragments, and incubated 15 min at 37 °C. Then, an excess of heparin (10 µg) or 300-fold molar excess of competing unlabeled DNA fragment were added. Samples were withdrawn and applied to a running 5% polyacrylamide gel after 0, 5, 10, 15, 30, 60, and 90 min of competitor addition. Quantification of the results were performed as above. In the case of MalR, the protein was used at 0.9 µM, and the competing unlabeled DNA was the M fragment.

Construction of pLS1mXGFP-- To change the DNA sequence of PX promoter at the -10 extended region, oligonucleotides mut1and mut2 were designed. The resulting sequence should change the wild type: 5'-TGTGCTATACT-3', into 5'-TGCACCCTACT-3' (changes in italics). Mutagenesis reactions were performed by PCR, using two pairs of oligonucleotides, mal3/mut1 and mal4/mut2, and pLS70 as DNA template. The PCR products obtained (80 and 474 bp) were purified, mixed in equimolecular amounts, denatured (95 °C, 5 min), and annealed (55 °C, 5 min). The overlapping products were extended with the Pfu enzyme (72 °C, 15 min), yielding a 516-bp DNA fragment, which includes the mutations in PX. This DNA fragment was next amplified using oligonucleotides mal3 and mal4, and the product was cleaved with EcoRI and XbaI. This DNA was used to replace the wild type fragment for the mutated one by cloning into pJDC9GFP (25). Finally, the EcoRI-ClaI fragment from the recombinant was cloned into the EcoRI-HindIII sites of plasmid pLS1 to obtain the desired plasmid, pLS1mXGFP. The entire nucleotide sequence of the region encompassing promoters, PM and PX, was determined with an automated sequencer equipment (Applied Biosystems 377) and the dye-deoxy termination procedure.

Measurement of GFP Activity-- Cells harboring plasmids (pLS1Er, pLS1GFP, pLS1MGFP, or pLS1mXGFP) were grown in medium containing 0.8% sucrose or maltose to middle exponential phase (OD650 of 0.4, about 3 × 108 colony-forming units/ml). Cells were pelleted by centrifugation and suspended in PBS buffer (10 mM Na2HPO4, 140 mM NaCl, 3 mM KCl), pH 7.2. Fluorescence was determined on a LS-50B spectrophotometer (PerkinElmer Life Sciences) by excitation at 488 nm and detection of emission at 510 nm. All experiments were performed at least three times. To measure GFP synthesis in a real-time scale, a procedure developed in our laboratory was followed (to be published elsewhere). Essentially, pneumococcal cells were grown as above in 0.8% sucrose-containing medium, harvested by centrifugation, and suspended at the same density in medium containing sucrose (0.8%) or maltose (5%). Aliquots (200 µl) of cells were used to follow fluorescence emission during various periods of time. As the control for background fluorescence, cells harboring plasmid pLS1Er were used, and their values were subtracted in each experiment.

    RESULTS AND DISCUSSION
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Transcription Initiation from Promoter PM-- Promoters PX and PM are placed in a divergent orientation within the noncoding intergenic region between operons malXCD and malMP (Fig. 1A). Hydroxyl radical interference assays showed that the MalR binding sites, the operators OX and OM, are placed just downstream of the promoters (22). The DNA sequence around PX (but not around PM) has a perfectly conserved "extended -10 region" (5'-TgTGcTATAcT-3') and a good -35 region (5'-TTGcaA-3'). Such an extension of the -10 region is commonly associated to strong promoters that may lack the -35 region both in S. pneumoniae (29) and E. coli (3). Initiation of transcription from PX (23) showed that the target of MalR protein at this promoter, the OX operator (22), was located downstream of the +1 start point (Fig. 1B).


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Fig. 1.   Organization of the S. pneumoniae mal regulon. A, schematic representation of the region, indicating: promoters (hatched lines), direction of mRNA synthesis (arrowheads), and coding regions (pointed rectangles). The negative regulatory role of MalR on promoters PX and PM is indicated. B and C, nucleotide sequence of the DNA region encompassing promoters PX (B) and PM (C). Relevant features of these regions are: promoters, with their -35 and -10 regions boxed, RNAP footprints (brackets), MalR operators (OX and OM, boxed), initiation of mRNAs for operons malXCD and malMP (transcription initiation sites indicated by arrows), and initiation codons and direction of synthesis (arrows). The putative UP sequences upstream of promoters PX and PM are also indicated. Oligonucleotides 1 and 2 used for PCR amplification (open arrows at the beginning and end of the sequence) are also shown.

To map the initiation of transcription from promoter PM, total mRNA was prepared from S. pneumoniae, and primer extension and endonuclease S1 assays were performed. Primer extension assays yielded a 130-nt protected band (Fig. 2A), whereas the major band observed by S1 mapping was 129 nt long (Fig. 2B). These results positioned the initiation of the malMP mRNA just at the beginning of the OM operator at this promoter (Fig. 1C). It was important to determine whether initiation of transcription of the malMP operon in both S. pneumoniae and E. coli occurred at the same position. Since the pneumococcal RNAP is not available to us, E. coli RNAP-directed in vitro transcription assays were carried out. As templates, the DNA fragments M (Fig. 2C) or XM (Fig. 3), were used. The former fragment contained only promoter PM, whereas the latter harbored both PM and PX. The results showed the synthesis of a run-off transcript of 130-131 nt (Fig. 2C), which placed the transcription initiation point from promoter PM at the same position than that obtained for S. pneumoniae. In the case of PX, in vitro transcription assays, using the X or the XM DNA fragments, showed synthesis of two run-off products of 110 and 112 nt (Fig. 3). The sizes of these transcripts are in accordance with the transcription initiation point previously determined for the malXCD operon in pneumococcal cells (23). We conclude that: (i) promoters PX and PM are functional in S. pneumoniae and E. coli, (ii) the promoters are equally recognized by both bacterial RNAP, and (iii) initiation of transcription takes place at the same position in both hosts. An interesting feature of the DNA regions upstream of promoters PX and PM, is the very high A+T-content (over 90%) from positions -40 to -65 (Fig. 1). These upstream regions share the consensus sequence found for UP elements in several promoters, both at their distal (AAA(a/t)(a/t)T(a/t)TTTT) and proximal (AAAA) regions (30), indicating that promoters PX and PM may have UP elements with which the alpha -subunit of RNAP would contact (3).


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Fig. 2.   Determination of the initiation start point of the malM mRNA by primer extension analyses (A) and by nuclease S1 mapping (B). The size of the region extended by reverse transcriptase was confirmed by run-off in vitro transcription assays (C). A, G, C, T, sequence reactions of the same region were performed as molecular size markers, using oligonucleotide 2 as the primer. Sizes (nt) of the products are indicated.


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Fig. 3.   Repression of mRNA synthesis by MalR protein. A, schematic representation of the DNA fragments used as templates, which carry promoter PX (fragment X), PM (fragment M), or both (fragment XM). B, in vitro run-off transcripts synthesized by the E. coli RNAP (22 nM) in the absence (0) or presence of increasing amounts of MalR (lane 1, 0.54; lane 2,1.08; lane 3, 1.62; lane 4, 2.16; lane 5, 2.70 µM protein). Both proteins were added simultaneously to the reaction mixtures. The sizes (nt) of the transcripts synthesized from promoters PM and PX are indicated to the left and right, respectively.

MalR Differentially Represses Transcription from Promoters PM and PX in Vitro-- Previous results obtained by transcriptional fusions between promoters PX and PM with two reporter genes indicated a preferential MalR repression on PM (22). To determine the degree of transcriptional repression by MalR protein on these promoters, direct measurements were performed by in vitro transcription assays. To this end, DNA fragments containing PX, PM, or both promoters (fragments X, M, or XM, respectively, as schematized in Fig. 3A) were used as templates to support RNAP-directed synthesis of run-off RNA products in the presence and absence of MalR. Reaction mixtures received increasing amounts of purified MalR protein, and transcription assays were initiated by addition of RNAP. The results showed that synthesis of the run-off transcripts (130-131 nt) from PM was strongly inhibited by MalR, even at the lowest protein concentration tested (Fig. 3B). The levels of MalR-mediated repression from PM were similar for both the M or the XM DNA fragments. In the case of promoter PX two main transcripts (110 and 112 nt) were synthesized, as expected from transcription initiating from the previously determined start point (23). However, MalR-mediated inhibition was only observed at the highest protein concentration (2.7 µM), a result found for both X or XM DNA fragments (Fig. 3B).

Quantification of the results indicated that full MalR-mediated repression of transcription from PM was achieved at protein concentrations above 1.62 µM, whereas repression from PX (at MalR concentrations of 2.7 µM) was at most 35% (fragment X) or 45% (fragment XM) of the value obtained in the absence of the repressor. In the absence of MalR, RNA synthesis from PM was about 6 times more efficient when the template carried only this promoter (fragment M) than when both promoters were present (fragment XM), which was not the case for transcription from PX (Fig. 3B). These findings suggested that RNAP may have a preferential recognition of promoter PX. If this were the case, transcription of the malMP operon could be reduced when transcription of the malXCD operon was fully functional, due to sequestering of RNAP. Thus, a delicate interplay between MalR and RNAP in the recognition of the promoters/operators sequences may take place, so that MalR-mediated repression would be much stronger on promoter PM than on PX, whereas RNAP recognition would be the opposite. Consequently, we can postulate that the main operator for the binding of MalR is OM, although the presence of auxiliary operators (like OX) could be required for maximum repression in vivo, as in the case of the LacI repressor and its operator sequences O1, O2, and O3 (31). Alternatively, existence of DNA sequences leading to promoter interference could also explain the above results (see below).

MalR repression was preferential on promoter PM, and the regions protected by the repressor were previously located downstream PM (22). To know the relative position of the RNAP-binding sites within the PM/OM region, the regions protected by RNAP were determined by DNase I footprinting assays, and these protected regions were compared with those generated by MalR. The results (not shown) indicated that RNAP covered ~69 nt on the promoter PM region, the footprints spanning from positions -47 to +22 (see Fig. 1C). This protection pattern is typical for most of the RPO complexes, and lies within the upstream limits found for short contacted areas and the normal downstream contact border (32). Bands showing hypersensitivity to DNase I cleavage were located at -37 and -45, plus an additional band at +18 (see Figs. 5, A and C). Appearance of enhanced bands at these positions could be due to the formation of a DNA bend at this region (see Ref. 5, and references therein). Consequently, we conclude that the regions protected by RNAP on promoter PM overlap with the OM operator.

Displacement of MalR by RNAP at Promoter PM-- To determine the differential affinity of MalR and RNAP for binding to promoter PM, we performed in vitro transcription experiments in the presence of increasing amounts of either protein, and using as template the M-DNA fragment (Fig. 3). In these assays, MalR or RNAP were first incubated with the template DNA prior to the addition of the competing protein to the reaction mixtures, and transcription was initiated by addition of the NTPs. When the competing protein was RNAP, it was apparent that the polymerase was able to displace the already bound MalR to its OM target (Fig. 4A) despite the long half-life of MalR-OM complexes (more than 90 min). Quantification of the results indicated that about 264 nM of RNAP were required to reach 90% of the synthesis obtained with RNAP alone (Fig. 4B). Thus, the rate of dissociation of the MalR-OM complex, in the presence of high concentrations of RNAP, would account for the frequency of transcription initiation from PM. When the converse experiments were performed, it was found that MalR was able to displace the RNAP already bound to PM (Fig. 4C), although transcription was reduced at most to about 40% in the presence of 2.7 µM of MalR (Fig. 4D). Comparison of these results with those obtained when RNAP and MalR were added simultaneously (Fig. 3B) showed that, at 1.08 µM MalR, transcription decreased only to 60% when RNAP was already bound to its target (Fig. 4D), whereas simultaneous addition of MalR and RNAP reduced transcription efficiency to 10% of the control (Fig. 3B, fragment M, lane 2). Thus, once a stable complex RNAP promoter is formed, displacement by MalR is inefficient, indicating that effective repression is exerted during the first steps of transcription. In the absence of MalR, RNAP would bind to promoter PM, generating mostly RPO stable complexes, productive for transcription, and only the unstable complexes would be susceptible to competition by MalR and, as a consequence, to repression.


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Fig. 4.   MalR-mediated inhibition of transcription from promoter PM. DNA protein complexes were allowed to form before addition of the competing protein, either RNAP (A and B) or MalR (C and D). A, MalR (2.16 µM) was added prior to the addition of the following concentrations of RNAP (nM): 0 (lane 0), 22 (lane 1), 44 (lane 2), 88 (lane 3), 132 (lane 4), 176 (lane 5), and 264 (lane 6) nM, (equivalent to 0, 0.16, 0.32, 0.64, 0.96, 1.28, and 1.92 units, respectively). B, quantification of the results of A. 100% is the value obtained in the absence of MalR. C, RNAP (22 nM) was added before the addition of the following amounts of MalR (µM): 0 (lane 0), 1.08 (lane 1), 2.16 (lane 2), 3.24 (lane 3), 4.32 (lane 4), and 5.4 (lane 5). D, quantification of the results. 100% is the value obtained in the absence of MalR. Arrows indicate the transcripts synthesized by RNAP. To quantify the percent of run-off transcripts (panels B and D), the bands with higher mobility were used.

Competition between MalR and RNAP for Promoter PM Occupancy-- The overlap of the RNAP- and MalR-induced footprints, and the in vitro transcription competition assays, suggested to us that the mechanism of MalR repression at PM could be due to competition between both proteins for promoter occupancy. To test this assumption, DNase I footprint and EMSA assays were performed in the presence of both proteins. In the DNase I footprint exclusion assay (Fig. 5), the XM-DNA fragment was terminally labeled in the coding strand of the malMP operon (Fig. 1B). RNAP (88 nM), and increasing amounts of MalR (0.54-1.62 µM) were added simultaneously, and the protection patterns were compared with those generated by MalR (Fig. 5B) or RNAP (Fig. 5C) alone. The results showed that the RNAP-generated footprints, and the characteristic RNAP-mediated hypersensitive bands disappeared as the MalR concentration was increased, demonstrating a progressive exclusion of the binding of RNAP to promoter PM (Fig. 5A). Similar results were obtained when the relative amounts of both proteins were reduced (data not shown). These results indicate that RNAP and MalR compete for the occupancy of the same sites within promoter PM, but do not demonstrate that both proteins bind to the same DNA molecules.


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Fig. 5.   Binding of MalR and of RNAP to promoter PM region assayed by protection against DNase I cleavage. DNase I footprinting analyses were carried out with the XM fragment, 5'-end-labeled in the malMP coding strand in the presence of RNAP (88 nM) and increasing amounts of MalR protein. A, lanes 0-3, 0, 0.54, 1.08, and 1.62 µM MalR; lane 4, DNase I protection pattern obtained with MalR alone. B, DNase I footprints generated by MalR on the same fragment. Lanes 0-2, 0, 0.54, and 1.08 µM MalR. The DNA sequence protected by MalR is indicated (boxed) to the right. C, DNase I footprints generated by RNAP on the same fragment. Lanes 0-3, 0, 18, 36, and 72 nM RNAP. Arrows point to hypersensitive bands generated by the binding of RNAP, and brackets show the extension of the footprints. A, G, C, T, nucleotide sequence of the same region.

The above question was addressed by characterization of ternary complexes generated by RNAP, MalR, and the DNA target. To this end, EMSA assays were done in which both proteins were added simultaneously to DNA fragments harboring PM (fragment M), PX (fragment X), or both promoters (fragment XM). When fragment X was tested (Fig. 6, panel X), retarded bands due to binding of MalR (bands M) or to RNAP (bands R) were distinguishable and, when both proteins were present, the specific complex generated by RNAP (36 nM) was almost insensitive to increasing amounts (0.22-0.9 µM) of MalR. This was not the case when fragment M was assayed (Fig. 6, panel M), since the intensity of the single band generated by RNAP bound to PM was reduced as the concentration of MalR in the assay was increased. These results indicated to us that RNAP binds preferentially to the PX/OX region, whereas MalR would bind preferentially to the region encompassing PM/OM.


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Fig. 6.   Competition between MalR and RNAP for binding to the promoter/operator regions measured by EMSA employing M, X, or XM DNA fragments. Both proteins were added simultaneously to the reaction mixtures. Concentrations of both proteins are indicated. The binding complexes with MalR or with RNAP are depicted as M or R, respectively (upper panels). In the lower panel, the retarded bands obtained for MalR or RNAP bound to one of the sites on the DNA fragment are denoted as M1 or R1, respectively; the complexes generated by MalR or RNAP to the two sites are indicated as M2 or R2, respectively. The band R/P indicates the binding of both MalR and RNAP to the same fragment. In all cases, F denotes the unbound DNA.

A more complex pattern was found when the DNA fragment employed contained both promoters (Fig. 6, panel XM). In this case, two retarded bands were detected when RNAP alone was used (bands R1 and R2). These bands may correspond to RNAP bound to one promoter (either PX or PM) or to both promoters. In that sense, when the amount of RNAP was increased, the RNAP-specific band of higher mobility (RNAP bound to PX or to PM; band R1) was shifted to the position of lower mobility, corresponding to the RNAP bound to both promoters (band R2). MalR protein alone generated also two retarded bands, corresponding to its binding to operator OM (for which it has higher affinity; band M1), and to both operators (OM and OX; band M2). Addition of increasing amounts of MalR to RNAP-DNA complexes showed the gradual disappearance of both RNAP-DNA complexes, and the appearance of a new complex migrating between both RNAP-promoter complexes (band R/P). We interpreted this latter band as corresponding to a ternary complex which involved RNAP bound mainly to promoter PX and MalR to the OM operator, within the same DNA molecules. To evaluate the involvement of MalR in the formation of these ternary complex, we took advantage of the inhibition of MalR-binding to DNA by maltose (22). We performed a similar EMSA assay with fragment XM but increasing amounts of maltose (from 0.35 to 5.6 mM) were added to the reaction mixtures. As expected, maltose gradually reduced the binding of MalR alone or in the presence of RNAP, so that the ternary MalR-RNAP-DNA complexes were decreased, this reduction being paralleled by a concomitant increase in the RNAP-DNA complexes (results not shown).

Taking the above results together, we may conclude that MalR represses transcription from promoter PM by binding to its operator OM, and hindering the binding of RNAP to its target, both proteins competing for the same DNA region. Competition between MalR and RNAP was more evident at the malMP operon than at the malXCD operon, because of the higher affinity of RNAP for PX. However, quantification of the half-lives of the RNAP-DNA complexes by challenging the complexes with an excess of competitor, showed no significant differences between the half-life of RNAP at PX (26 min) or at PM (25 min). This may reflect that sequences within or near the PX-OX DNA region may influence the binding of RNAP to its target DNA (see below).

Role of the OX Operator in the Level of MalR-mediated Repression of the malMP Operon-- Although MalR binds more weakly to operator OX than to OM, our results do not rule out that the former operator is required in vivo (in conjunction with OM) for either an optimal expression of the malMP operon or for effective repression, as shown for the LacI repressor (31). To test this hypothesis, transcriptional fusions were assayed in S. pneumoniae by placing the gene encoding GFP under the control of promoter PM. We used three plasmid constructions, namely pLS1GFP (25), pLS1mXGFP, and pLS1MGFP. Plasmid pLS1mXGFP carried a defective PX promoter in which the -10 region and the TG extension were altered. This mutation led to a 20-fold reduction in the binding of RNAP to the PX-OX DNA region, as compared with the wild type sequence (data not shown). In the case of plasmid pLS1MGFP, the sequence encompassing promoter PX was removed. As a control, plasmid pLS1Er (lacking the gfp gene) was used. Pneumococcal cells harboring plasmids were grown in media containing either sucrose (repressed conditions) or maltose (induced cultures) as carbon source, and the fluorescence of the cultures were measured. The values obtained (Table I) showed that the fluorescence of the maltose-induced cultures harboring pLS1GFP increased by a factor of about 6, as compared with the uninduced cells, a value that agrees with previous results (25). Mutations in the -10 region of PX (plasmid pLS1mXGFP) affected this value only slightly if at all. However, the ratio observed in the induced versus uninduced cultures increased nearly 12-fold in cells harboring pLS1MGFP (deletion of PX). Such an increase in fluorescence should be due to a 60% increase in the transcription rate from PM, demonstrating that the DNA region encompassing PX influenced negatively transcription from PM after induction. To test whether this increase was independent of the cell phase of growth, we measured the fluorescence of cells growing in microtiter plates (i.e. measurement of the synthesis of GFP in real time). The results showed that cells harboring plasmids with a deleted PX responded to induction with higher levels of GFP synthesis than the wild type (Fig. 7), demonstrating that more than promoter PX itself, there is a DNA region proximal to it, which is responsible for the interference with transcription from PM. Curiously, deletion of the PX promoter region led to a reduction in the basal levels of GFP synthesis observed in the uninduced cultures. This difference could be due to a weak promoter activity, within the deleted region, reading in orientation toward promoter PM. Although we have not found indications of the existence of such a putative promoter, the presence of an extremely A+T-rich region makes it possible to find several sequences that resemble -10 regions. The existence of a weak promoter in this region was previously suggested (26).

                              
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Table I
Determination of fluorescence in pneumococcal cells harboring plasmids
Bacterial cultures (S. pneumoniae R61) were grown in media containing sucrose (S) or maltose (M) to an OD650 of 0.8 (about 7 × 108 cfu/ml) with selective pressure (erythromycin, 1 µg/ml). Cells (1 ml from each culture) were sedimented by centrifugation and suspended in the same volume of phosphate-buffered saline. Aliquots (200 µl) were used to measure the fluorescence in microtiter plates. The fluorescence intensity were obtained by subtraction of the values obtained in the isogenic strain with pLS1Er. Three independent clones were used to determine each value. S.D. values for all assays did not exceed 10%.


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Fig. 7.   Expression of GFP in cells of S. pneumoniae as a function of the time of growth. Data were collected every 10 min, and fluorescence was detected in media containing maltose (open symbols) or sucrose (closed symbols). Plasmids used were pLS1GFP, containing both promoters (open circle , ), pLS1MGFP, harboring a deletion in promoter PX (triangle , black-triangle), and pLS1mXGFP( , black-square), containing the wild type promoter PM and the mutated promoter PX.

Taking the above results together, we propose that the different affinities of RNAP and MalR for Ox and OM may play a role in the induction of both promoters so that, under non-induced conditions, MalR would bind preferentially to OM, leading to a basal expression from PX. When the system is induced by maltose, promoters PM and PX would compete for RNAP binding, with RNAP having a higher affinity for PX than for PM. An alternative explanation for our results would be that an interference between PX and PM may occur. This phenomenon is well characterized in phage lambda  promoters PR and PRM, which have start sites separated by 83 phosphodiester bonds (33). However, in the PX and PM pneumococcal promoters, such a distance is of 424 phosphodiester bonds. So far, we have been unable to show experimentally the possible existence of a MalR-mediated DNA loop (not shown), which would bring about both promoters shortening this distance.

We conclude that MalR represses transcription at promoter PM at an early stage, prior to RPO formation, most likely by binding to the OM operator and hindering the access of RNAP to the promoter. Two observations support the hypothesis that there is a greater exclusion between MalR and RNAP at promoter PM than at PX. First, the position of OM within the malMP operon is such that it almost overlaps with the +1 position, which is not the case for the OX operator. Second, the MalR footprints at the PM/OM DNA region are totally included within those generated by RNAP. In vivo and in vitro observations indicate that there is a differential and opposite affinity of MalR and RNAP for the promoter/operator regions of the pneumococcal malMP and malXCD operons (22). In addition, measurements of amylomaltase (the product of gene malM) activity in pneumococcal cells showed a 20-fold induction by maltose when the operon was in a single copy (24), whereas partial de-repression was found when the cells harbored multiple copies of the malR gene (21). The implications of these findings are that the malMP operon, involved in the metabolism of maltodextrins, should be shut off in the absence of the inducer, whereas the malXCD, involved in the uptake processes, would be functioning, at least at a basal level, in all growth conditions. In this sense, the -10 extension of promoter PX could account for a rather high basal level of transcription. As a consequence, pneumococcal cells would always be ready for the uptake of nutrients, whereas their metabolism would be operative only when needed. Thus, regulation of the two operons may depend on the interplay between the relative affinities of MalR and RNAP for their operator/promoter regions.

    ACKNOWLEDGEMENTS

We thank members of M. Espinosa's laboratory for helpful discussions, S. Adhya for criticisms, and C. Rosenow for communication of results prior publication. We acknowledge the technical help of M. T. Alda.

    FOOTNOTES

* This work was supported by Comisión Interministerial de Ciencia y Tecnología Grant BMC2000-0550, by Comunidad Autónoma de Madrid Grant 07B/30/99, and by the Program for Strategic Groups.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to the late Eladio Viñuela, a great scientist and a friend.

To whom correspondence should be addressed. Tel. 34-91-5611800; Fax: 34-91-5627518; E-mail: mespinosa@cib.csic.es.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010911200

    ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; bp, base pair(s); EMSA, electrophoretic mobility shift assay; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Er, erythromycin, GFP, green fluorescent protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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