From the 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
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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.
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 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.
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
[ 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 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 [ 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 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.
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
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 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 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.
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.
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.
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
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
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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP and polynucleotide kinase (28).
80° °C. No loss of DNA binding activity was observed during 1-year storage.
- 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.
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.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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.
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
-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.
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.
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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.
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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.
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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.
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).
Determination of fluorescence in pneumococcal cells harboring
plasmids
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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 ( ,
), pLS1MGFP, harboring a deletion in
promoter PX (
,
), and pLS1mXGFP(
,
), containing the wild type promoter PM and
the mutated promoter PX.
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.
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.
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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.
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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
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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.
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