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Journal of Bacteriology, November 1998, p. 5727-5732, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transcriptional Regulation of the
Streptococcus mutans gal Operon by the GalR
Repressor
Dragana
Ajdi
and
Joseph J.
Ferretti*
Department of Microbiology and Immunology,
The University of Oklahoma Health Sciences Center, Oklahoma City,
Oklahoma 73104
Received 1 June 1998/Accepted 26 August 1998
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ABSTRACT |
The galactose operon of Streptococcus mutans is
transcriptionally regulated by a repressor protein (GalR) encoded by
the galR gene, which is divergently oriented from the
structural genes of the gal operon. To study the regulatory
function of GalR, we partially purified the protein and examined its
DNA binding activity by gel mobility shift and DNase I footprinting
experiments. The protein specifically bound to the
galR-galK intergenic region at an operator sequence, the
position of which would suggest that GalR plays a role in the
regulation of the gal operon as well as
autoregulation. To further examine this hypothesis, transcriptional start sites of the gal operon and the
galR gene were determined. Primer extension analysis showed
that both promoters overlap the operator, indicating that GalR most
likely represses transcription initiation of both promoters. Finally,
the results from in vitro binding experiments with potential effector
molecules suggest that galactose is a true intracellular inducer of the
galactose operon.
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INTRODUCTION |
Streptococcus mutans is
the major causative agent of dental caries, and sugar metabolism is
known to play an important role in causing this disease. S. mutans possesses different mechanisms for the utilization of
sugars, and recently the operon involved in galactose
metabolism via the Leloir pathway has been cloned and characterized
(2). The transcription of the structural genes
(galK, galT and galE) comprising the
gal operon is repressed in the absence of galactose
and is subject to catabolite repression in the presence of glucose
(2). The galR gene of S. mutans has been shown to specify a repressor of the galactose operon; unlike in Escherichia coli and Streptomyces
lividans, this gene is located immediately upstream and is
divergently oriented from the structural genes (see Fig. 8)
(2). Computer analysis has shown that GalR belongs to the
GalR-LacI family of transcriptional regulators that bind as a dimer to
the specific DNA sequence (16, 26, 27).
To study the regulation of the S. mutans gal
operon at the molecular level, GalR was partially purified and
used in gel mobility shift and footprinting assays. In this report, we
demonstrate that transcriptional regulation of the gal
operon of S. mutans is mediated by a protein
product of the galR gene (GalR). In the absence of
galactose, GalR binds to a palindromic sequence which overlaps the
galR and gal operon promoters and
probably represses their initiation of transcription.
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MATERIALS AND METHODS |
Growth conditions.
E. coli strains were grown in LB or
M9 medium (20) supplemented with appropriate antibiotics
(ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; and rifampin, 200 µg/ml). S. mutans strains were grown in semidefined
medium (18, 24) supplemented with either galactose or
glucose and kanamycin (400 µg/ml) when necessary.
DNA manipulations and sequencing.
Protocols for plasmid
extraction, digestion of DNA with restriction enzymes, gel purification
of DNA fragments, DNA ligation, and agarose and polyacrylamide gel
electrophoresis have been described elsewhere (20).
Sequencing reactions were done with a Sequenase version 2.0 kit
(U.S. Biochemical) according to the manufacturer's protocol.
Overexpression of galR.
Plasmid pSF813, used for
overexpression of galR, was constructed by cloning a 1.8-kb
HindIII-EcoRI fragment, which contains the
galR gene and its own translation signals (2),
into the pT7T318U expression vector (Pharmacia), thereby positioning
galR under the control of the T7 promoter. A second plasmid,
pGP1-2, was used as a source of the T7 RNA polymerase gene,
whose expression is under the control of a temperature-sensitive 
repressor cI857) (22).
The E. coli expression strain (JM109 transformed with pSF813
and pGP1-2) and control strain (JM109 transformed with pT7T318U and
pGP1-2) were grown to an A600 of 0.5 to 0.6 in 1 ml of M9 medium supplemented with ampicillin, kanamycin, 0.05 mM
thiamine and 0.1% Casamino Acids. The culture was then incubated at
42°C for 30 min, then rifampin was added, and incubation continued for 20 min at 42°C. Cells were pelleted, washed twice with M9 medium,
resuspended in labeling medium (M9 supplemented with ampicillin, kanamycin, 0.05 mM thiamine, and rifampin), and incubated at 42°C for
10 min. After addition of 20 µCi of
L-[35S]methionine-cysteine (ICN), the cells
were incubated for 2 h at 30°C, pelleted, washed twice with
Tris-EDTA, and resuspended in loading buffer (0.08 M Tris [pH 6.8],
0.1 M dithiothreitol, 2% sodium dodecyl sulfate [SDS], 10%
glycerol, 0.1 mg of bromphenol blue per ml). The cells were then
denatured at 95°C for 5 min, and the proteins were analyzed on an
SDS-12% polyacrylamide gel.
Partial purification of GalR.
E. coli JT34
[F
Strr his relA1
galR(B-C)::Cmr]
(25), transformed with plasmids pGP1-2 and pSF813, was grown
in 500 ml of LB medium supplemented with ampicillin (200 µg/ml) and
kanamycin to an A600 of 0.5. galR was
induced by increasing the culture temperature to 42°C for 45 min, and
the cells were grown at 37°C for another 4 h in the presence of
rifampin. The cells were harvested by centrifugation, washed two times
in buffer A (20 mM Tris-HCl [pH 7.5]), suspended in lysis buffer (20 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1 mM
phenylmethylsulfonyl fluoride, DNase I [1 µg/ml], RNase I [1
µg/ml]) (15), and lysed by use of a French press (100 MPa). Cell debris was removed by consecutive centrifugation at
15,000 × g for 10 min followed by 60 min at
100,000 × g. The supernatant was removed, and
approximately 15 mg of total proteins was loaded on a 9-ml heparin
column (Affi-Gel heparin gel; Bio-Rad) that had been equilibrated and
washed with buffer A. Proteins were eluted with a 0.2 to 1.2 M NaCl
gradient made in buffer A. The 1 M NaCl fraction, which contained most
of the GalR protein, was concentrated by centrifugation in Centriprep
10 and Centricon 10 (Amicon) concentrators. The control protein extract
from JT34 transformed with vectors only (pT7T318U and pGP1-2) was
isolated by the same procedure. The protein concentration was
determined by using a Pierce protein determination kit with bovine
serum albumin as a standard. The N-terminal amino acid sequence of GalR was determined by the Molecular Biology Resource Facility of the University of Oklahoma Health Sciences Center.
Gel mobility shift assay.
The probe used for the gel
mobility shift assay was prepared by digesting plasmid pSF806
(2) with EcoRI and XbaI to give a
200-bp fragment. This fragment was separated on a 5% polyacrylamide gel, electroeluted, and end labeled with [
-32P]ATP
(6,000 Ci/mmol) and T4 kinase after dephosphorylation (20). The radiolabeled probe (5 to 10 ng) was mixed with different
concentrations of partially purified GalR (32.5 to 227.5 ng) in binding
buffer [25 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM
CaCl2, 2 mM dithiothreitol, 50 µg of bovine serum albumin
per ml, 50 mM KCl, 25 µg of poly(dI-dC)-poly(dI-dC) per ml] in a
total volume of 20 µl. Sugars were added to the reaction mixture 5 min before radiolabeled probe was added, at a final concentration of
0.5% (28 mM), when indicated. Incubation was carried out for 15 min at
room temperature. The DNA-protein complex was separated from the
unbound DNA fragment on a 5% native polyacrylamide gel, using 1×
Tris-borate-EDTA (20) as the electrophoresis buffer. D-(+)-Galactose (SigmaUltra; catalog no. G-6404) and other
sugars were purchased from Sigma.
DNase I footprinting analysis.
For DNase I footprinting, the
468-bp EcoRI-PstI DNA fragment from pSF806
(2) was electroeluted from a 5% polyacrylamide gel and
labeled at the 3' end with [
-32P]dATP (800 Ci/mmol)
via the large fragment of DNA polymerase I (20). The
reaction mixture contained the same components as the mixture used for
the gel mobility shift assay, except that the concentrations of protein
extract were 0.5 to 3 µg and the concentrations of the DNA fragments
were 50 to 80 ng in a total volume of 40 µl. Incubation was carried
out for 45 min at room temperature. This was followed by digestion with
20 mU of DNase I (Sigma) in an appropriate buffer (200 mM Tris-HCl [pH
8.3], 500 mM KCl, 20 mM MgCl2) for 2 min at room
temperature. The reaction was stopped with 20 µl of 25 mM EDTA,
ethanol precipitated, and resuspended in a sequencing loading buffer.
DNA fragments were separated on a 6% sequencing gel.
Site-directed mutagenesis.
Site-directed mutagenesis of the
GalR-binding sequence was performed by using an Altered Sites II in
vitro mutagenesis system kit (Promega) as recommended by the
manufacturer. The mutagenic oligonucleotide was
5'-ATCTGAGGTCAATATGGATCCTAAAATTTTACTAA-3' (positions 
28 to 62 relative to the gal
operon transcriptional start site). The control mutagenic
oligonucleotide was
5'-GATAATGGCTACATTAGGATCCATTGCAAAATTAGC-3' (positions 115 to 150 relative to the gal
operon transcriptional start site). The underlined sequences of
five nucleotides replaced the wild-type sequences (TAAAA and TCTTT,
respectively). Fragments that carry the altered operator sequences were
purified and end labeled as described above.
RNA isolation.
Total cellular RNA was isolated from an
S. mutans LT11 (23) exponential-phase
culture by the hot acidic phenol method (14), with
modifications. Lysis of the cells was accomplished by a Mini Beadbeater
(Biospec Products, Inc.) using zirconia-silica beads (0.1-mm diameter).
The concentration of RNA was determined by A260
measurements, and the quality of RNA was analyzed on a conventional Tris-borate-ethidium bromide agarose gel (12).
Primer extension.
To determine the transcriptional start
site of the galR and gal operons,
oligonucleotides 5'-CATCTTTGTTCAATACTC (positions +127 to
+144 relative to the galR transcriptional start site) and
5'-GTAGCGTCTGCTTCTCTTCC (positions +68 to +88 relative to the gal operon transcriptional start site),
respectively, were used. Primer extension analysis was performed by
using avian myeloblastosis virus reverse transcriptase as described
previously (11).
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RESULTS |
Overproduction and partial purification of GalR.
Because most
cellular regulatory proteins are present at a relatively low level, we
have overexpressed the galR gene in E. coli. galR
was placed under the control of the T7 promoter of the pT7T318U vector,
forming pSF813. This plasmid was then introduced into a strain
containing a second plasmid, pGP1-2, that carries the T7 RNA
polymerase gene under the control of a temperature-sensitive repressor (22). Expression of galR in a
strain containing both plasmids was activated by increasing the
temperature to 42°C, resulting in derepression of the T7 RNA
polymerase and consequently transcription and translation of
the galR as well as 
-lactamase, since there are no
terminators on the pT7T318U vector. The same E. coli host
transformed with pT7T318U and pGP1-2 was used as a control, and a
comparable experiment was performed. Rifampin was used to inhibit the
E. coli RNA polymerase of both strains. The proteins
observed by autoradiography of an SDS-polyacrylamide gel, after
induction and labeling with [35S]methionine, are shown
in Fig. 1. Two proteins of similar
molecular weight (MW) were radioactively labeled in the induced control strain (Fig. 1, lane 1), whereas two different-MW proteins were visible
in the induced galR expression strain (Fig. 1, lane 3). A 29-kDa protein is -lactamase precursor, whereas a 27-kDa protein is processed -lactamase (22). A 37-kDa protein was
present in the induced galR expression strain only (Fig. 1,
lane 3), and it migrated with a mobility which corresponded to that
predicted from the deduced amino acid sequence of GalR (2).
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FIG. 1.
Autoradiograph showing overexpression of galR
in E. coli. Lane 1, control protein extract of E. coli cells harboring pT7T318U and pGP1-2 (the culture was induced
at 42°C); lane 2, control protein extract of E. coli cells
harboring pT7T318U and pGP1-2 (the culture was not induced [30°C]);
lane 3, protein extracts of E. coli cells harboring
expression plasmid pSF813 (galR in pT7T318U) and pGP1-2 (the
culture was induced at 42°C); lane 4, protein extracts of E. coli cells harboring expression plasmid pSF813 (galR in
pT7T318U) and pGP1-2 (the culture was not induced [30°C]). Rifampin
was added in all samples except the one in lane 4. Since a prestained
protein MW standard was used, positions of the bands were marked on the
autoradiograph according to their positions on the polyacrylamide
gel.
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Previous experiments had shown that GalR from
S. mutans
could be successfully produced in
E. coli; thus, it was
decided to
overexpress and partially purify the product of
galR starting
with a 500-ml culture of the
galR
expression strain (as described
in Materials and Methods). Many
DNA-binding proteins show affinity
for heparin-agarose, and in
this study we used heparin chromatography
for partial purification
of GalR. Fractions of the protein extract
eluted from a
heparin-agarose column with an NaCl gradient were
analyzed on
an SDS-polyacrylamide gel. The major band visible
in the 1 M NaCl
fraction, which migrated as a 37-kDa protein (Fig.
2), was sequenced. Nine amino acids of
this protein, obtained
by N-terminal protein sequencing, were identical
to the deduced
amino acid sequence of GalR. This partially purified
GalR was
then used for in vitro DNA binding experiments.
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FIG. 2.
Partially purified GalR. A 1 M NaCl protein fraction was
analyzed on an SDS-12% polyacrylamide gel after chromatography on a
heparin-agarose column. Proteins in the gel were detected with
Coomassie blue staining (20). Lane 1, MW protein standard;
lane 2, partially purified GalR protein extract.
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Specific binding of GalR to the gal operon
promoter region.
The DNA binding activity of the partially
purified GalR was determined in a gel mobility shift assay. As shown in
Fig. 3A, the mobility of the specific
200-bp DNA fragment that contained the galR-galK
intergenic region was shifted upon addition of the partially purified
GalR. The fraction of retarded DNA fragments increased as the
protein concentration increased. Only one shifted band was
observed in the autoradiograph if the protein concentration added to
the reaction mixture was in the range of 32.5 to 227.5 ng/reaction,
indicating that one DNA-protein complex was formed.
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FIG. 3.
Specific binding of GalR to the galR-galK
intergenic region. Autoradiographs of a gel mobility shift assay on a
5% polyacrylamide gel are shown. Approximately 10 ng of the specific
(200-bp) end-labeled DNA fragment that contained galR and
gal operon promoters and different, partially
purified protein extracts were used in each assay. (A) Titration gel
mobility shift assay using partially purified GalR protein extract.
Concentrations of the protein extract: lane 1, 0 ng; lane 2, 32.5 ng;
lane 3, 65 ng; lane 4, 97.5 ng; lane 5, 130 ng; lane 6, 162.5 ng; lane
7, 195 ng; lane 8, 227.5 ng. (B) Competition gel mobility shift assay.
The concentration of the identical but unlabeled specific DNA fragment
(added 5 min before labeled DNA) was approximately 20 ng per reaction.
The unlabeled fragment was not added in lane 6. Concentrations of
partially purified GalR protein extract: lane 1, 0 ng; lane 2, 32.5 ng;
lane 3, 97.5 ng; lane 4, 162.5 ng; lane 5, 227.5 ng; lane 6, 97.5 ng.
(C) Control gel mobility shift assay using control protein extract
partially purified from the E. coli host carrying the vector
without a galR insert. Concentrations of the protein
extract: lane 1, 0 ng; lane 2, 97.5 ng; lane 3, 162.5 ng; lane 4, 227.5 ng.
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To further analyze the binding of the GalR protein extract, competition
experiments were performed. Binding was almost completely
abolished if
a specific competitor (20 ng of unlabeled identical
DNA fragment) was
added to the reaction mixture 5 min before addition
of labeled DNA (10 ng) (Fig.
3B), whereas an excess amount (0.5
to 1 µg) of a
nonspecific competitor, poly(dI-dC)-poly(dI-dC),
that was routinely
used in gel mobility shift experiments had
no effect on binding.
Furthermore, when the gel mobility shift
assay was performed with a
protein extract prepared from the culture
that contained a vector with
no insert, a specific DNA-protein
complex was not formed (Fig.
3C), suggesting specific binding
of GalR to the
galR-galK intergenic region. GalR binding was not
observed with DNA fragments either upstream (0.4 kb) or downstream
(0.25 kb) of the specific DNA fragment or with the fragment carrying
the
galT-galE intergenic region (results not shown).
Effect of D-galactose on repressor binding.
To
determine if different sugars play a role in GalR binding to the
specific DNA fragment, we performed gel mobility shift experiments using different sugars in each reaction mixture (galactose, glucose, maltose, fructose, melibiose, raffinose, lactose,
glucose-1,6-biphosphate, fructose-1,6-biphosphate, and all
intermediates of the Leloir pathway for galactose metabolism
[galactose-1-phosphate, UDP galactose, UDP glucose,
glucose-1-phosphate, and glucose-6-phosphate]). Addition of
D-galactose, the inducer of the gal
operon (2), in the reaction mixture almost
completely abolished binding of partially purified GalR to the
specific DNA fragment (Fig. 4,
lanes 1 to 4), suggesting that D-galactose is a true
intracellular inducer of the gal operon. None of the
other sugars had an effect on GalR binding.
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FIG. 4.
Autoradiograph of a gel mobility shift assay showing the
effect of D-galactose on repressor binding. Specific
end-labeled DNA fragment (approximately 10 ng) was mixed with different
amounts of the GalR protein extract, with (lanes 1 to 4) or without
(lanes 5 to 9) D-galactose (which was added 5 min before
the labeled probe in a final concentration of 0.5%). Free DNA
fragments were separated from the DNA-protein complex on a 5%
polyacrylamide gel. Concentrations of partially purified GalR: lanes 1 and 6, 250 ng; lanes 2 and 7, 180 ng; lanes 3 and 8, 110 ng; lanes 4 and 9, 40 ng; lane 5, 0 ng.
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Identification of repressor-binding region.
The location of
the repressor-binding site was identified by a DNase I footprinting
assay using the partially purified GalR and an end-labeled 468-bp DNA
fragment that contained the galR-galK intergenic region and
5' end of the galR gene. The protected region extended from
35 to 70 starting from the transcriptional start site of the first
structural gene (galK) of the gal operon
(Fig. 5). Several hypersensitive sites,
between positions 90 and 120, were visible on the autoradiograph
upon addition of the protein extract and DNase I digestion, suggesting
a major conformational change of substrate DNA upon repressor binding.
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FIG. 5.
DNase I footprinting of GalR. The DNA fragments were
separated on a 6.5% sequencing gel. Concentrations of partially
purified GalR: lane 1, 0 µg; lane 2, 3 µg; lane 3, 1.5 µg; lane
4, 1 µg; lane 5, 0.75 µg. The amount of the specific, 468-bp
3'-labeled DNA fragment was approximately 50 ng/reaction. Nucleotide
positions are given on the left relative to the galK
transcriptional start site.
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It is known that DNA-binding proteins often bind to regions in which
the nucleotide sequence displays dyad symmetry. Sequence
analysis of
the protected region revealed an imperfect palindrome
(see Fig.
8). To
further examine the same region, a 5-bp change
(TAAAA to GATCC;
positions
43 to
47) in the right half of the
palindrome was created
by site-directed mutagenesis. As a control,
a 5-bp change (TCTTT to
GATCC; positions
130 to
134) was created
outside the detected
palindromic sequence, and both mutations
were verified by DNA
sequencing. When the mutated operator was
used as a probe for a
gel mobility shift assay, formation of a
DNA-protein complex was
decreased considerably (Fig.
6, lanes
1 to 4), whereas the control mutation had no effect on DNA-protein
binding (Fig.
6, lanes 5 to 8), suggesting that the detected
protected
region is the true operator of the
gal
operon.
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FIG. 6.
Effects of mutations in the operator region on formation
of the DNA-protein complex. The DNA fragment with mutations in the
operator region was used for gel mobility shift assays in lanes 1 to 4;
the DNA fragment that carries mutations outside of the operator region
was used for gel mobility shift assays in lanes 5 to 8; nonmutated DNA
fragment was used for gel mobility shift assays in lanes 9 to 12. Concentrations of the partially purified GalR: lanes 1, 5, and 9, 0 ng;
lanes 2, 6, and 10, 130 ng; lanes 3, 7, and 11, 162.5 ng; lanes 4, 8, and 12, 195 ng.
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Determination of the gal operon and
galR transcriptional start sites.
The orientation of
the regulatory and structural genes of the gal
operon suggested that their transcription originated within the
galR-galK intergenic region. Several attempts to detect a galR transcriptional start site have failed, most likely
because of the low level of specific mRNA present. To enrich the amount of mRNA, galR was cloned into shuttle vector pDL276
(7), and S. mutans LT11 was transformed with
this construct. Total RNA was then isolated, and a primer extension
experiment was performed. Two extension products, with 5' ends mapping
at a location 68 and 69 nucleotides upstream of the galR
translational start site, were detected (Fig.
7A), and so the exact transcription
initiation site remains conjectural. No product was detected after
primer extension analysis performed with total RNA isolated from
S. mutans LT11 carrying a vector without the insert
(data not shown). The galR transcriptional start site
revealed that the galR promoter overlaps the gal
operon promoter and that its 10 region overlaps the operator
(Fig. 8).To determine a transcriptional start point of the gal
operon structural genes, total RNA was isolated from S. mutans LT11 cultures grown in semidefined medium
supplemented with galactose, and primer extension analysis was
performed with a specific primer. The 5' end of the mRNA for the
gal operon was located 25 nucleotides upstream of
the galK translational start site (Fig. 7B), in agreement
with the location of the promoter predicted from the
nucleotide sequence. The 35 region of this promoter overlaps
with the operator sequence (Fig. 8).
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FIG. 7.
Primer extension analysis of galR and
gal operon transcription in S. mutans. Total RNA from S. mutans LT11 cells grown
in semidefined medium supplemented with galactose was isolated, and
approximately 20 µg of total RNA was used for the primer extension
reactions. Sequencing reactions were prepared with the same primer used
for primer extension analysis. (A) Lane 1, determination of the 5' end
of the galR transcript; (B) lane 2, determination of the 5'
end of the gal operon transcript.
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FIG. 8.
S. mutans gal operon and
nucleotide sequence of the galR-galK intergenic region. (A)
galR, galactose repressor gene; galK,
galactokinase gene; galT, galactose-1-phosphate
uridyltransferase gene; galE, UDP glucose-4-epimerase gene.
Arrows show orientation of transcription of the genes; open boxes
represent probes used in gel mobility shift (probe 1) and footprinting
(probe 2) assays. (B) The sequence protected by GalR from DNase I
digestion is in boldface; putative promoters are underlined.
Predictions for 10 and 35 promoter regions were based on the
gram-positive organism promoter consensus sequence (9)
and/or the promoter consensus sequence for the genus
Streptococcus (17). The operator region mutated
by site-directed mutagenesis is boxed; the region of dyad symmetry is
indicated by horizontal arrows. RBS stands for ribosome binding site;
vertical arrows indicate the transcriptional start sites. Numbers below
and above the sequence refer to galR and galK
genes, respectively.
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DISCUSSION |
Sequence analysis of GalR has shown that this protein possesses a
helix-turn-helix motif at its N terminus, which is typical for
DNA-binding proteins (16, 26, 27). Additionally, it has been
shown that mutation in galR leads to constitutive expression of galK, the first structural gene of the S. mutans gal operon, which suggests that GalR is a
repressor of the galactose operon (2). To verify
that GalR indeed regulates the genes responsible for metabolism of
galactose via protein-DNA interaction, GalR was overproduced in
E. coli, partially purified, and used in an in vitro binding
study. Gel mobility shift, DNase I footprinting, and site-directed
mutagenesis analyses showed that GalR binds to the galR-galK
intergenic region at a position that is equidistant from the two genes.
Nucleotide sequence analysis of the repressor-protected region
revealed that it spans an imperfect inverted repeat. The dyad
symmetry of the repressor-protected region and the presence of a
dimerization region at the C-terminal part of the GalR amino acid
sequence suggests that this repressor binds to its
cis-active sequence as a dimer, where each monomer
interacts with one half of the palindrome. Furthermore,
site-specific mutagenesis of the right half of the operator
considerably decreased but did not completely abolish binding of GalR
to its operator in vitro, which might mean that the GalR dimer was
still able to partially interact with the nonmutated half of the
operator through one monomer.
Our previous results showed that the most effective inducer of the
gal operon was galactose itself (2),
although a high level of induction was also obtained with raffinose, a
galactose-containing carbohydrate. The uptake of raffinose eventually
results in an increase in galactose concentration in the cell, since
the galactose moiety of raffinose is released intracellularly by
-galactosidase. To determine whether galactose is a bona fide
intracellular inducer, gel mobility shift experiments were repeated in
the presence of different sugars. Our observation that galactose
abolishes binding of GalR, whereas none of the other tested sugars,
including intermediates of the Leloir pathway, had no effect on
DNA-protein interaction, strongly suggests that galactose is the true
intracellular inducer of the gal operon.
Primer extension analysis of the galR and the gal
operon transcriptional start site has shown that there are two
divergent, overlapping promoters in the galR-galK intergenic
region. One of these promoters is responsible for galR
transcription, whereas the other enables transcription of the
structural genes of the operon. As shown in Fig. 8, the 10
promoter region of galR and part of the 35 region of the
gal operon promoter overlap the operator, strongly
suggesting that GalR, if bound to this operator, interferes with
transcription from both promoters. Previous analyses have shown that
GalR negatively regulates expression of the gal operon. It has also been shown that several repressors
negatively regulate their own expression (10, 19, 21, 29).
To study further the nature of GalR autoregulation, primer extension
experiments were done under maximal inducing and repressing conditions;
the same products were detected, although the level of transcription of
galR was higher in galactose-grown cells. These results, as well as the overlapping position of the gal operator and
galR promoter, indicate that GalR probably negatively
regulates its own synthesis. To our knowledge, this is the first study
demonstrating negative autoregulation of galR transcription.
Our results also suggest that there is a basal level of gal
operon transcription when glucose is present in the growth
medium, which correlates with previous results of galactokinase assays
(2). It has been shown that UDP galactose 4-epimerase (GalE)
is required for glycosylation and cell wall synthesis in E. coli (1, 6). Even if galactose is not present in the
growth medium, UDP galactose is generated from UDP glucose by GalE,
which is necessary for galactosyl lipid synthesis. In order to fulfill
the same requirement, galE of S. lividans
must be transcribed from its constitutive promoter located immediately
upstream of the gene (8). Analysis of the chemical composition of S. mutans purified cell walls revealed
that galactose is one of the components (28), and GalE might
also be necessary for cell wall synthesis in this organism. Since we
did not observe promoter activity in the galT-galE
intergenic region under tested conditions (data not shown), the basal
level of transcription from the gal operon promoter
might be necessary to fulfill a requirement for constitutive
galE expression.
Our results indicate that the structural organization and
regulation of the gal operon of S. mutans are different from those in E. coli and
S. lividans. The genes comprising the E. coli
gal operon are transcribed from two overlapping promoters
(P1 and P2) which are negatively regulated by the galactose repressor (GalR) (1, 6). GalR binds as a dimer to two operator sites located outside the promoters. Repression of the gal
operon promoters requires the presence of histone-like HU
protein, which mediates loop formation through contact of the GalR
dimers bound to the operators (3, 4, 13). A similar
situation is observed in S. lividans, where the genes
responsible for galactose utilization, as in E. coli,
are organized in a polycistronic manner, and two identified
promoters (galP1 and galP2) have been shown to be
independently regulated (8). galP1, which is
located immediately upstream of the operon, is induced in the
presence of galactose, whereas galP2, the internal promoter
located upstream of the galE gene, is responsible for
constitutive expression of the galE and galT genes. Transcription of the S. mutans gal
operon is driven by a single promoter located upstream of
galK, the first structural gene. This promoter
sequence overlaps a single operator, and evidence presented
here indicates that binding of the S. mutans GalR to the operator represses RNA polymerase binding to the
gal operon promoter, although it cannot be ruled out
that both GalR and RNA polymerase are able to bind
simultaneously. GalR would then inhibit a step of initiation subsequent
to polymerase binding through GalR-RNA polymerase
interactions. Furthermore, the same operator overlaps the
galR promoter, which, unlike the case for E. coli, suggests dual function of the GalR: regulation of the
gal operon transcription as well as autoregulation.
These studies enabled us to formulate a simple model for regulation of
the galactose operon by the gal repressor. When
galactose is not present in the growth medium, binding of
GalR to its operator represses initiation of transcription from both
promoters. Addition of galactose inactivates the repressor, allowing
transcription to proceed. It has been known that steric hindrance could
occur between RNA polymerase molecules attempting to bind
simultaneously to the overlapping promoters (5).
This competition could reduce the activity of one promoter (in this
case probably the galR promoter) and increase the activity
of the other. Indeed, our results showed that the gal
operon promoter is about 10 times stronger than the galR promoter (unpublished data). This type of control might
be necessary for expression of the gal operon genes
in order for their products to be present in a fixed molar ratio. When
the concentration of galactose decreases, GalR molecules are able to
bind their operator again. This model does not exclude the possibility
that another protein directly or indirectly affects regulation of the
gal operon. It has been shown recently that, besides
the Gal repressor, the histone-like protein HU is required for
transcriptional regulation of the gal operon in
E. coli (3, 4, 13). Whether such a protein is
also required for transcriptional regulation of the S. mutans gal operon remains to be determined.
| |
ACKNOWLEDGMENTS |
This investigation was supported by USPHS research grant
DE08191 from the National Institutes of Health.
We acknowledge W. M. McShan and R. E. McLaughlin for helpful
discussions and review of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, The University of Oklahoma, HSC, 940, S. L. Young Blvd., Oklahoma City, OK 73104. Phone: (405)
271-2332. Fax: (405) 271-3151. E-mail:
Joe-Ferretti{at}uokhsc.edu.
| |
REFERENCES |
| 1.
|
Adhya, S.
1987.
The galactose operon, p. 1503-1512.
In
F. C. Neidhardt, L. J. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C.
|
| 2.
|
Ajdic, D.,
I. C. Sutcliffe,
R. R. B. Russell, and J. J. Ferretti.
1996.
Organization and nucleotide sequence of the Streptococcus mutans galactose operon.
Gene
180:137-144[Medline].
|
| 3.
|
Aki, T., and S. Adhya.
1997.
Repressor induced site-specific binding of HU for transcriptional regulation.
EMBO J.
16:3666-3674[Medline].
|
| 4.
|
Aki, T.,
H. E. Choy, and S. Adhya.
1996.
Histone-like protein HU as a specific transcriptional regulator: co-factor role in repression of gal transcription by GAL repressor.
Genes Cells
1:179-188[Abstract].
|
| 5.
|
Beck, C. F., and R. A. J. Warren.
1988.
Divergent promoters, a common form of gene organization.
Microbiol. Rev.
52:318-326[Free Full Text].
|
| 6.
|
de Crombrugghe, B., and I. Pastan.
1980.
Cyclic AMP, the cyclic AMP receptor protein, and the dual control of the galactose operon, p. 303-324.
In
J. H. Miller, and W. S. Reznikoff (ed.), The operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 7.
|
Dunny, G. M.,
L. N. Lee, and D. J. LeBlanc.
1991.
Improved electroporation and cloning vector system for gram-positive bacteria.
Appl. Environ. Microbiol.
57:1194-1201[Abstract/Free Full Text].
|
| 8.
|
Fornwald, J. A.,
F. J. Schmidt,
C. W. Adams,
M. Rosenberg, and A. M. Brawner.
1987.
Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon.
Proc. Natl. Acad. Sci. USA
84:2130-2134[Abstract/Free Full Text].
|
| 9.
|
Graves, M. C., and J. C. Rabinowitz.
1986.
In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene.
J. Biol. Chem.
261:11409-11415[Abstract/Free Full Text].
|
| 10.
|
Gunsalus, R. P., and C. Yanofsky.
1980.
Nucleotide sequence and expression of E. coli trpR, the structural gene for the trp aporepressor.
Proc. Natl. Acad. Sci. USA
77:7117-7121[Abstract/Free Full Text].
|
| 11.
|
Ivanisevic, R.,
M. Milic,
D. Ajdic,
J. Rakonjac, and D. J. Savic.
1995.
Nucleotide sequence, mutational analysis, transcriptional start site, and product analysis of nov, the gene which affects Escherichia coli K-12 resistance to the gyrase inhibitor novobiocin.
J. Bacteriol.
177:1766-1771[Abstract/Free Full Text].
|
| 12.
|
Lunsford, R. D.
1995.
Recovery of RNA from oral streptococci.
BioTechniques
18:412-413[Medline].
|
| 13.
|
Lyubchenko, Y. L.,
L. S. Shlyakhtenko,
T. Aki, and S. Adhya.
1997.
Atomic force microscopic demonstration of DNA looping by GalR and HU.
Nucleic Acids Res.
25:873-876[Abstract/Free Full Text].
|
| 14.
|
Magni, C.,
P. Marini, and D. de Mendoza.
1995.
Extraction of RNA from Gram-positive bacteria.
BioTechniques
19:882-884.
|
| 15.
|
Mohr, C. D.,
N. S. Hibler, and V. Deretic.
1991.
AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site.
J. Bacteriol.
173:5136-5143[Abstract/Free Full Text].
|
| 16.
|
Nguyen, C. C., and M. H. Saier.
1995.
Phylogenetic, structural and functional analyses of the LacI-GalR family of bacterial transcription factors.
FEBS Lett.
377:98-102[Medline].
|
| 17.
|
Podbielski, A.,
J. A. Peterson, and P. Cleary.
1992.
Surface protein-CAT reporter fusions demonstrate differential gene expression in the vir regulon of S. pyogenes.
Mol. Microbiol.
6:2253-2265[Medline].
|
| 18.
|
Russell, R. R. B.
1979.
Purification of Streptococcus mutans glucosyltransferase by polyethylene glycol precipitation.
FEMS Microbiol. Lett.
6:197-199.
|
| 19.
|
Saint-Girons, I.,
N. Duchange,
G. N. Cohen, and M. M. Zakin.
1984.
Structure and autoregulation of the metJ regulatory gene in E. coli.
J. Biol. Chem.
259:14282-14285[Abstract/Free Full Text].
|
| 20.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 21.
|
Schell, M. A.
1993.
Molecular biology of the LysR family of transcriptional regulators.
Annu. Rev. Microbiol.
47:597-626[Medline].
|
| 22.
|
Tabor, S., and C. C. Richardson.
1985.
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.
Proc. Natl. Acad. Sci. USA
82:1074-1078[Abstract/Free Full Text].
|
| 23.
|
Tao, L.,
T. J. MacAlister, and J. M. Tanzer.
1993.
Transformation efficiency of EMS-induced mutants of S. mutans of altered cell shape.
J. Dent. Res.
72:1032-1039[Abstract/Free Full Text].
|
| 24.
|
Terleckyj, B.,
N. P. Willett, and G. D. Shockman.
1975.
Growth of several cariogenic strains of oral streptococci in a chemically defined medium.
Infect. Immun.
11:649-655[Abstract/Free Full Text].
|
| 25.
|
Tokeson, G. P.,
E. Garger, and S. Adhya.
1991.
Further inducibility of a constitutive system: ultrainduction of the gal operon.
J. Bacteriol.
173:2319-2327[Abstract/Free Full Text].
|
| 26.
|
Weickert, M. J., and S. Adhya.
1992.
A family of bacterial regulators homologous to gal and lac repressors.
J. Biol. Chem.
267:15869-15874[Abstract/Free Full Text].
|
| 27.
|
Weickert, M. J., and S. Adhya.
1992.
Isorepressor of the gal regulon in Escherichia coli.
J. Mol. Biol.
226:69-83[Medline].
|
| 28.
|
Wetherell, J. J. R., and A. S. Bleiweis.
1978.
Antigens of Streptococcus mutans: isolation of a serotype-specific and a cross-reactive antigen from walls of strain V-100 (serotype e).
Infect. Immun.
19:160-169[Abstract/Free Full Text].
|
| 29.
|
Wilson, R. L., and G. V. Stauffer.
1994.
DNA sequence and characterization of GcvA, a LysR family regulatory protein for the Escherichia coli glycine cleavage enzyme system.
J. Bacteriol.
176:2862-2868[Abstract/Free Full Text].
|
Journal of Bacteriology, November 1998, p. 5727-5732, Vol. 180, No. 21
0021-9193/98/$04.00+0
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Abstract
Introduction
