From the Division of Cellular, Molecular and Microbial Biology, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Received for publication, October 29, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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Glucitol induction in Bacillus
subtilis requires a transcription activator, GutR, and a sequence
located upstream of the gut promoter. To understand the
initial steps involved in the GutR-mediated transcription activation
process and the physiological roles of glucitol, GutR was overproduced
and purified. In the absence of glucitol, GutR exists as a monomer and
binds directly to its binding site in the gut regulatory
region. This binding site was mapped to a 29-base pair imperfect
inverted repeat located between With the addition of glucitol to Bacillus subtilis, a
set of glucitol-inducible genes is selectively turned on (1). These inducible genes include gutA- and gutB-encoding
glucitol permease and glucitol dehydrogenase, respectively. They are
the members of the gut (glucitol
utilization) operon and are arranged in the order from
gutB to gutA. This operon is subject to both
negative and positive regulations at the transcription level with the
positive regulation playing a major role in the induction process. The regulatory region of the gut operon can be divided into
three parts (2). The central portion is an unusual promoter with its Several different motifs can be found in GutR, including a typical
helix-turn-helix motif for DNA binding at the N-terminal region, motifs
A and B for a nucleotide binding site, and tetratricopeptide repeats at the C-terminal region (4). To understand the
regulatory mechanism of glucitol induction in B. subtilis,
we report in this study the overproduction and purification of GutR.
This protein indeed binds specifically to the regulatory region
upstream of the gut promoter, and there is only a single
GutR binding site in that region. The precise boundary of this binding
site was mapped. Interestingly, GutR can form a complex with its
binding site even in the absence of glucitol. However, in the presence of glucitol, the real-time kinetic measurement with a BIAcoreX biosensor demonstrates that GutR binds extremely tightly to its binding
site and that this tight binding is the result of a change in the
off-rate (dissociation rate constant) of the binding reaction. Molecules other than glucitol are unable to induce a similar effect. Possible steps involved in the initial stage of the GutR-mediated transcription are discussed.
Construction of the GutR Expression Vector--
The expression
vector used in this study is the B. subtilis pUB18-P43
vector (5). This vector is a pUB110 derivative with a kanamycin
resistance marker and a strong, constitutively expressed B. subtilis promoter P43 to drive the transcription of genes of interest inserted in the polylinker region located downstream of the
promoter. gutR was obtained from pU1 (4), which carries the
chromosomal gutR region by ScaI and
BamHI digestion. The resulting gutR gene was
inserted into the polylinker region of SmaI/BamHI double-digested Escherichia coli Bluescript vector
(pBS) to generate pBSGUTR. The digestion of pBSGUTR by
KpnI and BamHI allowed the release of the
gutR fragment that could be inserted into the
KpnI- and BamHI-digested pUB18-P43 vector to
generate pUB18P43GUTR.
Expression and Purification of GutR--
B. subtilis
WB600 is a six-extracellular protease-deficient strain that is used
here routinely for expression study (6). WB600(pUB18P43GUTR) and the
negative control strain WB600(pUB18P43) were cultivated in
superrich medium (7) with kanamycin at a level of 10 µg/ml at
37 °C. Cells were harvested at different time points. Both total and
fractionated cellular proteins (soluble and insoluble) were
analyzed by 12% SDS-polyacrylamide gel electrophoresis (PAGE). For
GutR purification, WB600(pUB18P43GUTR) was cultivated under the
above-mentioned conditions for 8 h with a cell density around 800 Klett units, and cells were collected by centrifugation for 10 min at 7000 × g. Cell pellets from a 500-ml culture
were resuspended in 40 ml of Buffer A (50 mM Tris-HCl, pH
8.0, 5 mM EDTA, and 1 mM phenylmethylsulfonyl
fluoride) and lysed by French press (10,000 p.s.i. with three or
four passes). Cell debris was removed by centrifugation at 27,000 × g for 20 min at 4 °C. The soluble fraction was applied
through a DEAE-52 cellulose column (2.5 × 40 cm) equilibrated
with Buffer A, followed by washing the column with 4 column volumes of
buffer until a value of A280 of 0.005 or less
was achieved. The column was then eluted with a linear NaCl gradient
(0-0.5 M) in Buffer A. Fractions were analyzed by
SDS-PAGE, and the GutR-containing fractions were pooled and dialyzed extensively with several changes in Buffer A. The dialyzed fraction was applied to a heparin-Sepharose column (3 ml of bed volume). After washing with a 2-column volume of Buffer A to lower the
value of A280 to 0.005, the column was eluted
with a linear NaCl gradient (0-0.5 M) in Buffer A. Fractions containing pure GutR were pooled and stored at
Molecular Weight Determination--
To determine the molecular
weight of GutR in solution, a Superose 6 HR column (Amersham Pharmacia
Biotech) was used in the Bio-Rad Biologic work station (Bio-Rad). The
column was calibrated with a molecular weight marker kit (Amersham
Pharmacia Biotech). Kav is defined as
(Ve Gel Mobility Shift Assay and in Vivo Mapping of GutR Binding
Site--
Specific DNA fragments for gel mobility shift assays were
generated by PCR and labeled at the 5' ends with T4 polynucleotide kinase and [ Generation of Biotinylated DNA Fragment by PCR for Kinetic
Assays--
For GutR binding studies with the BIAcoreX biosensor, a
DNA fragment containing the sequence from Immobilization of Streptavidin to Sensor Chips--
To
immobilize biotinylated DNA fragments to the sensor chips to study GutR
interaction with its target site, streptavidin has to be immobilized to
the carboxymethyl dextran on the CM5 sensor chip (research grade)
(BIAcore Inc., New Jersey) with an amine coupling kit from BIAcore Inc.
The coupling reaction was performed according to the following steps.
After mixing an equal volume of 100 mM
N-hydroxysuccinimide and 400 mM
N-ethyl-N'-(dimethylaminopropyl)carbodiimide together, 30 µl of this mixture was injected to activate the surface of the sensor chip. Streptavidin at a concentration of 30 µg/ml was
then injected. In the BIAcoreX system, the chip can be divided into two
flow cells. Each flow cell had appropriately 2250 resonance units of
streptavidin immobilized (~2.25 ng/mm2). Excess reactive
groups on the chip surface were then deactivated by the injection of 30 µl of 1 M ethanolamine. To eliminate any noncovalently
bound streptavidin from the chip, several injections of 10 µl of
0.05% SDS were performed, and the residual SDS molecules were then
removed by several washes with TEN buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% surfactant P20).
Determination of the Association and Dissociation Rate Constants
of GutR with Its Target DNA Using BIAcoreX
Biosensor--
Biotinylated polylinker sequence was immobilized to the
first flow cell at a final quantity of 84 resonance units to serve as a
negative control for GutR binding. The biotinylated GutR binding
site-containing fragment was immobilized in the second flow cell to 71 resonance units. With this level of the GutR binding site
immobilized, no mass transport effect that would interfere with the
kinetic measurement was observed (10). GutR in a binding buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05%
surfactant P20, and 20 mM MgCl2) was injected
into the sensor chip and passed from the first flow cell to the second
flow cell. The binding of GutR was monitored by the change of resonance
units with time. The on-rate (association rate constant,
ka) was determined via a two-step analysis.
In the first step, the binding rate, expressed in terms of
dR/dt (where R represents the
resonance unit), was plotted against the response (R). Based
on the kinetic equation dR/dt = Other Methods--
Plasmid isolation from and DNA transformation
to E. coli and B. subtilis, DNA subcloning, PCR
amplification, and SDS-PAGE were performed as described previously (2).
The amounts of purified GutR were determined by measuring the
absorbance at 280 nm under denaturing conditions as described by Gill
and von Hippel (11). The molar extinction coefficient at
A280 of 149,550 M Overproduction of GutR--
gutR was inserted
into a high copy number B. subtilis plasmid pUB18-P43
with the strong and constitutively expressed B. subtilis P43
promoter installed in the vector to drive the transcription of
gutR. The resulting vector, pUB18P43GUTR, was
transformed to a six-extracellular protease-deficient B. subtilis strain WB600, and the transformants were found to
overproduce and stably accumulate a 95-kDa protein with the molecular
mass agreeable with the calculated molecular mass (95,076) of GutR.
This protein was stable in the intracellular environment at least up to
12 h after inoculation (Fig.
1A). Fractionation studies
indicated that more than 95% of this protein was in the soluble
intracellular fraction (data not shown). Overproduction of GutR in
E. coli using T7 RNA polymerase systems (14, 15) was not
successful. The lack of GutR overproduction in E. coli is
not attributable to the presence of rare arginine codons in
gutR because the use of strains that overproduce rare arginine tRNAs does not improve the situation (16).
Development of a Two-step Scheme for GutR Purification--
The
isoelectric point of GutR was estimated to be 6.1. By adjusting the pH
to 8.0, GutR should be negatively charged and would be able to bind to
the positively charged DEAE-cellulose (DE52). Therefore, the first step
in this purification scheme was loading the cell lysate from
WB600(pUB18P43GUTR) to a DE52 column (Fig. 1B). The bound
GutR protein was eluted with a linear salt gradient from 0 to 0.5 M NaCl. This step served three purposes. First, it
could concentrate GutR from the crude cell lysate into a few fractions
that were rich in GutR. Second, chromosomal DNA could be separated from
GutR because GutR was eluted from the column at the salt concentration
of 0.15 M whereas chromosomal DNA was eluted from the
column at a salt concentration of 0.45 M. Third, many cellular proteins could also be separated from GutR. Fractions containing GutR were pooled and dialyzed to remove salt. The sample was
then applied to a heparin-agarose column based on the assumption that
GutR is a DNA-binding protein and should have an affinity to heparin.
Indeed, this step was very effective in removing other contaminating
proteins (Fig. 1B). The eluted GutR protein was estimated to
be at least 95% pure. The identity of this purified protein as GutR
was confirmed by N-terminal sequencing of the first five amino acid
residues of this protein. Approximately 4-6 mg of purified GutR can be
obtained from one liter of cell culture. Because there were not that
many endogenous 95-kDa proteins in the crude extract, purification of
GutR was monitored by analyzing the presence of the 95-kDa protein in
the eluted fractions. With the purification of GutR and the generation
of GutR-specific antibodies, subsequent rounds of GutR purification
were monitored by Western blot.
GutR Exists as a Monomer in Solution--
To determine whether
GutR exists as a monomer or an oligomer in solution in the absence of
glucitol, the molecular mass of purified GutR was determined via gel
filtration. GutR has an apparent molecular mass of 115 kDa (data not
shown). Because this value is much smaller than that expected for a
dimeric GutR, the result suggests that GutR exists as a monomer in
solution and is likely to be a slightly elongated molecule rather than
a globular molecule. The same apparent molecular mass was obtained when
tested with a wide range of GutR concentrations (0.5-5
µM). Because the highest intracellular GutR concentration
under the induced condition was estimated to be less than 1 µM based on the Western blot analysis, GutR in the
cellular environment in the absence of glucitol should exist as monomers.
GutR Binds Specifically to a Sequence Located Upstream of the
Promoter of the gut Operon in a Glucitol-independent
Manner--
Previous studies indicated that the sequence upstream of
the gut promoter is required for the expression of the
gut operon (2) and that GutR is the transcription activator
that is hypothesized to bind to the upstream regulatory sequence (4).
To examine this possibility, a gel mobility shift experiment was
performed with two PCR-amplified, 5' end-labeled fragments. One
fragment had the sequence corresponding to the In Vitro Deletion Mapping of the GutR Binding Site--
Examining
the upstream region of the gut promoter, a 29-bp imperfect
inverted repeat sequence located from positions In Vivo Deletion Mapping of the GutR Binding Site--
To
strengthen the two conclusions concerning the GutR binding site in the
gut regulatory region, fragments L1 to L4 were individually inserted in front of a promoterless lacZ reporter gene in
the B. subtilis integration vector pDH32. The resulting
gut-lacZ transcriptional fusions were integrated at the
amyE locus in the chromosome. As shown in Fig.
4, integrants with fragments L1-L3
carrying the intact inverted repeat sequence showed comparable
Glucitol Induces a Tight Binding of GutR to Its Binding
Site--
In vivo studies indicate that glucitol serves as
an inducer to turn on the gut operon in B. subtilis. However, the roles of glucitol in the GutR-mediated
transcription activation are unknown. To explore the possible
functional roles of glucitol in this induction process, the rate
constants for both the binding and dissociation reactions of GutR to
its binding site in the absence and presence of glucitol were
determined using the BIAcoreX biosensor. In this study, a GutR
binding site containing a fragment ( Transcriptional regulation of glucitol induction in B. subtilis is mainly regulated via transcription activation. GutR
serves as a transcription activator in this process (4), and a region upstream of the gut promoter is also required for induction
(2). It is logical to predict that GutR binds to this upstream
regulatory region and activates transcription. In this study, with
overproduction and purification of GutR, GutR was demonstrated to bind
directly to the upstream region of the gut promoter. This
binding did not require the presence of glucitol. The GutR binding site
was mapped to the 29-bp imperfect inverted repeat located at positions
With GutR as the transcription activator for the gut operon,
another logical prediction is that GutR should serve as the sensor for
the presence of the inducer (glucitol) of this system. Isolation of a
gutR1 mutant that results in the constitutive expression of
the gut operon even in the absence of glucitol supports this idea (1). In this mutant, a C to A change at the nucleotide level leads
to the substitution of Ser-289 by an arginine in GutR (4). In the
present study using a biosensor with purified GutR, the idea for
glucitol to bind directly to GutR is further strengthened. In the
presence of glucitol, GutR binds extremely tightly to its target site.
The on-rate of the binding reaction is not significantly changed,
whereas the off-rate is significantly reduced to a degree that cannot
be measured by the biosensor system. This effect is glucitol-specific.
If glucitol was replaced by either 2% xylitol or 2% glucose in the
binding reactions, the dissociation rate constants under these
conditions were comparable with those observed for GutR in the absence
of any ligand. These data suggest that glucitol can bind to GutR and
trigger GutR to have an extremely tight binding to its target site,
whereas xylitol and glucose are unable to bind to GutR. This implies
that glucitol is the true inducer for this system.
In many activator-dependent operons, activators gain the
ability to bind to the target sites only in the presence of a specific ligand such as catabolite activator protein with cAMP (19). Alternatively, activators can bind to the target site with equal affinity no matter whether a specific ligand is present or not as
observed for SoxR, an activator that activates genes in response to
oxidative stress. However, only the Fe-SoxR but not the apo-SoxR activator can stimulate transcription (20). In the case of GutR, it has a relatively tight binding to its target site
(Kd = 9 × 10 A tight binding can be achieved by an increase in the on-rate, a
decrease in the off-rate, or a combination of both. The on-rate is a
second order reaction governed mainly by diffusion and long range
electrostatic interactions. In contrast, the off-rate is a first order
reaction determined by many short range interactions between the two
interacting molecules, such as hydrogen bonding and hydrophobic
interactions. In the case of GutR, only the off-rate of the
GutR binding reaction in the presence of glucitol is affected. This can
be achieved by at least three possible mechanisms. 1) The binding of
glucitol to GutR can induce GutR to have conformational changes so that
more residues in the DNA binding region of GutR are available to make
extensive contacts with the GutR binding site. 2) Besides the
helix-turn-helix region, extra contacts can possibly be made between
GutR and the sequences flanking the GutR binding site via DNA wrapping.
DNA wrapping around the hexameric arginine repressor has been suggested
to account for the tight binding of the arginine repressor to its
operator (22). 3) Glucitol may induce GutR to oligomerize. The presence
of multiple DNA binding domains in these oligomers will make sure that
the GutR binding site is always in contact with some of the GutR DNA
binding domains in the GutR oligomer.
Although the sequence search of GutR did not find any known regulatory
proteins that show significant homology to GutR, this protein shares
some similarities to the properties of the members of the 100-kDa
transcription activator family, such as MalT (23, 24) and AcoK (25).
They all are transcription activators with a molecular mass in the
range of 100 kDa and there is a nucleotide binding site in each of
these transcription activators. They activate promoters recognized by
the major RNA polymerase (i.e. the
E78 and
50, and there is only one
GutR binding site within the regulatory region. The kinetic parameters
of the interaction between GutR and its binding site were monitored in
real time using surface plasmon resonance. The half-life of the
GutR-DNA complex in the absence of glucitol was estimated to be 6.8 min. In contrast, in the presence of glucitol, the half-life of the
complex was extended to longer than 19 h by affecting only the
off-rate but not the on-rate. This effect is glucitol-specific. These
data indicate that glucitol binds to GutR and induces GutR to have an
extremely tight binding at its binding site. The physiological relevance of this process in transcription activation is discussed.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 and
35 elements similar to those recognized by the major RNA
polymerase (E
A) separated from each other by
a 15-bp1 spacer rather than a
typical 17-bp spacer. An inverted repeat sequence located downstream of
the promoter serves as a negative regulatory element (2). Although
deletion of this sequence does not result in the constitutive
expression of the gut operon in the absence of glucitol, the
induced expression from this system increases by almost 4-fold. A
region (from
126 to
48 with the transcription start site as +1)
located upstream of the promoter is required for glucitol induction and
is suggested to contain binding sites for a transcription activator,
GutR. The structural gene encoding GutR is located upstream of the
gut operon and is transcribed in an opposite direction
relative to that of the gut operon (3, 4). Inactivation of
this gene abolishes the glucitol induction of the gut operon.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
V0)/(Vt
V0). Vt is the total
bed volume of the column (24 ml). V0 is the void
volume of the column, and using blue dextran, it was determined
to be 6.4 ml. Ve is defined as the volume at which
the protein elutes.
-32P]ATP as described previously
(8). The boundary for these fragments was specified in Fig. 3. An
EcoRI site was introduced at the 5' end of these fragments,
and a BamHI site was introduced at the 3' end of these
fragments. DNA binding reactions were performed in a total volume of 30 µl in a binding buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM dithiothreitol, 5 mM EDTA, 3% Ficoll, 1 µg of sonicated salmon sperm DNA,
a 6 nM DNA probe, and 0-15 nM purified GutR.
The reaction mixtures were incubated at room temperature for 15 min and
then loaded onto a 6% polyacrylamide gel in 0.5 × TBE
buffer (45 mM Tris-borate, pH 8.0, 1 mM EDTA). The gel was run at 175 V in 0.5 × TBE buffer for 3 h and
then dried for autoradiography. Some of these fragments were also
inserted in front of the promoterless lacZ in pDH32 (9), a
B. subtilis integration vector, to generate transcriptional
fusions. The integration of pDH32 derivatives to chromosomal DNA and
selection of integrants were performed as described by Ye and Wong (2).
The
-galactosidase activity from integrants was assayed in the
presence and absence of glucitol, and the activity was expressed in
Miller units (2).
93 to
15 of the
gut operon was amplified using PCR with a pair of primers,
GUTB-93F (5'-GGGAATTCCAGCGTTTTTTGTATATG-3') and GUTB-15B
(5'-GGGGATCCTGCTTTTTAACACGTTCAAC-3'). The 5' end of the GUTB-15B primer
was biotinylated. This sequence carries the GutR binding site. To have
a negative control without a GutR binding site, the polylinker region
from pBS was amplified using universal (5'-GTAAAACGGCCAGT-3') and
reverse universal (5'-GTCCTTTGTCGATACTG-3') primers. The 5' end of the
universal primer was also biotinylated. PCR amplification
resulted in the generation of a 164-bp DNA fragment carrying a
sequence from the polylinker region of pBS. These primers were ordered
from the University Core DNA Services at the University of Calgary.
(kaC + kd)R + kaCRmax (10), such a plot
should yield a straight line with the slope (designated as
ks) representing the value of
kaC + kd
(i.e. ks = kaC + kd). In the
second step, by plotting the ks values generated with different concentrations (C, 1.78-52 nM)
of GutR, a straight line was generated, and the slope of this line
represented ka. At the end of the injection, the
GutR sample was replaced by the injection of binding buffer, and the
dissociation of GutR was monitored for 5 to 15 min. The dissociation
rate constant (off-rate, kd) was determined based on
the following equation,
ln(R0/Rt) = kd(t
t0) (10).
By plotting ln(R0/Rt) versus (t
t0), the
value of kd was determined. To examine the effect of
glucitol, xylitol, and glucose on GutR binding to its target DNA, each
of these components was added to the binding buffer at a final
concentration of 2%, and the effect of individual components on GutR
binding was monitored. All of these measurements were performed at
25 °C.
1
cm
1 was used to estimate the amounts of GutR. DNA
concentration was determined as described by Sambrook et al.
(12). N-terminal sequencing of purified GutR was performed as described
by Ng et al. (13).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overproduction and purification of GutR.
A, the expression profile of GutR from WB600(pUBP43GUTR)
analyzed by SDS-PAGE. M, molecular weight markers.
1, sample from the control strain WB600(pUB18) collected
11 h after inoculation. 2-8, samples from the GutR
overproduction strain WB600(pUBP43GUTR) collected 4, 6, 8, 9, 10, 11, and 12 h after inoculation, respectively. The amount of the total
intracellular proteins loaded in each lane is normalized against the
cell density. The dot marks the GutR protein band.
B, a SDS-PAGE profile showing GutR purification at various
stages. M, molecular weight markers. 1, sample
from the crude extract of WB600(pUBP43GUTR). 2 and
3, pooled GutR-containing fractions after DEAE-52 and
heparin-agarose columns, respectively.
132 to
14 region
from the regulatory region of the gut operon with the
transcription start site at +1. The other fragment was a 168-bp
fragment amplified from the polylinker region of the E. coli
Bluescript plasmid (pBS) to serve as a negative control. In the
presence of excess nonlabeled sonicated salmon sperm DNA, each of these
labeled fragments was incubated with an increasing concentration of
GutR from 5 to 50 nM. As shown in Fig.
2, GutR bound specifically to the
regulatory region of the gut operon but not to the
polylinker region from the Bluescript vector. With increasing
concentrations of GutR, there was a corresponding increase for the
GutR-DNA complex. This binding did not require the presence of
glucitol.
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Fig. 2.
GutR interacts specifically with its target
sequence. Lanes 1-6, gel retardation analysis of the 5'
end-labeled DNA fragment ( 132 to
14) carrying the GutR binding site
with increasing concentrations of GutR (0, 5, 15, 30, 40, and 50 nM, respectively). Lanes 7-12, the DNA fragment
containing the polylinker region from pBS was used in the binding assay
with the same concentration of GutR as specified in lanes
1-6. All these fragments were generated by PCR. 6 nM
end-labeled fragment was used in each assay. F, the position
of the free DNA. B, the position of the GutR-DNA complex. A
minor band presented in lanes 1-6 may represent the
presence of a low level of labeled GutR-containing DNA
fragments in its single-stranded form.
78 to
50 is likely
to be the GutR binding site (Fig.
3A). Because this sequence is
highly AT-rich and the entire regulatory region of the gut
operon is also AT-rich, it would be important to determine both the
number and the location of the GutR binding site within this region.
Gel mobility shift assays were performed using two series of DNA
fragments carrying the regulatory region of the gut operon
for the study. These DNA fragments were systematically shortened either
at the left or the right arm (Fig. 3B). For fragments with
deletion at the left arm, three deletions were made. Fragments L1 to L3
contained the intact inverted repeat sequence. In fragment L3, all of
the nucleotide sequence upstream of this inverted repeat was deleted.
In fragment L4, half of the inverted repeat sequence in the left arm
was also deleted. For the right arm deletion series, fragments R1 to R3
contained the intact inverted repeat whereas fragment R4 had the right
arm of the inverted repeat sequence deleted. Mobility shift assays with
GutR concentration ranging from 1 to 15 nM demonstrated
that DNA fragments (fragments L1 to L3 and R1 to R3) carrying the
intact inverted repeat could be selectively retarded by GutR (Fig.
3C). The concentrations of GutR required to shift 50% of
these fragments ([GutR]50) were in the range 7-10
nM (Fig. 3D). DNA fragments containing either the left (fragment L4) or the right (fragment R4) half of the inverted repeat sequence were either not retarded at all or only weakly retarded by GutR at the concentration range 1-15
nM. These data have three implications. First, the
29-bp inverted repeat serves as the GutR binding site. Second, there is
only one GutR binding site in the region tested (from
143 to +1)
because all sequences upstream and downstream of this inverted repeat
could be deleted without affecting the binding affinity of GutR to
these fragments. Furthermore, only one retarded complex could be
observed in all these mobility shift studies. These observations
strengthened the idea that there was a single GutR binding site in the
gut operon regulatory region. The last implication is that
GutR should bind to its binding site in the form of a dimer or a higher
oligomer because GutR could form an extremely weak complex with the
sequence that covered only half of the intact GutR binding site.
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Fig. 3.
In vitro deletion mapping of the
GutR binding site. A, sequence of the regulatory region
of the gut operon. +1, the transcription
start site. B, two series of fragments generated for the
in vitro GutR binding study. L1-L4,
fragments with deletions in the left arm. R1-R4,
fragments with deletions in the right arm. C, gel mobility
shift studies by GutR using fragments shown in B. In these
binding reactions, increasing GutR concentrations were used (0, 1, 3, 8, 12, and 15 nM). D, estimation of the
concentration of GutR required to shift 50% of the labeled DNA
fragments.
-galactosidase activities (1428-1609 Miller units) under the
induction condition. In the absence of glucitol, only a background
level (2-3 Miller units) of
-galactosidase activity was observed.
These observations indicate that the sequence located between
143 and
79 is not required for the glucitol-inducible expression. Consistent
with the in vitro observations, the integrant with fragment
L4, which had the left half of the inverted repeat deleted, showed the
expression at the basal level. This indicates that the inverted repeat
sequence is indeed required for glucitol induction.
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Fig. 4.
In vivo mapping of the GutR
binding site. DNA fragments carrying different lengths of the
gut regulatory region were inserted upstream of a
promoterless lacZ reporter gene in an integration vector,
pDH32, to create gutB-lacZ fusions. Each of these
integration vectors was integrated at the amyE locus in the
B. subtilis 168 genome. Integrants I-III carry the intact
GutR binding site, whereas integrant IV has only half of the intact
GutR binding site. -Galactosidase activity was expressed in Miller
units (2). amyE, the
-amylase gene locus in the B. subtilis genome. F and B, the front and back
portions of the
-amylase gene, respectively. +1, the
transcription start site of the gut operon. Black
box, the location of the 29-bp inverted repeat that represents the
GutR binding site.
95 to
15) with the nucleotide
at position
15 biotinylated was generated by PCR amplification and
unidirectionally immobilized to biosensor chips that had been coupled
with streptavidin. The GutR binding site located at the distal end of
the immobilized fragment would be available for GutR binding. The
sequence between
49 and
15 served as a spacer between the site of
immobilization and the GutR binding site. Fig.
5A showed the plots of
ks versus GutR concentration, and the slopes
of these plots represent the association rate constants. With the plots
of ln(R0/Rt) versus
time, the slopes of these lines represent the dissociation rate
constants (Fig. 5B). As shown in Table
I, GutR indeed could bind to its target
site in the absence of glucitol as observed with the gel mobility shift
assay. Interestingly, although the association rate constants of GutR
to its binding site in the presence or absence of glucitol were almost
the same, there was a significant difference for the dissociation rate
constants under these two different conditions. In the presence of
glucitol, GutR bound to the target site so tightly that the
dissociation rate constant was beyond the limit (1 × 10
5 s
1) that could be measured by the
BIAcoreX biosensor. This effect was glucitol-specific (Table I).
View larger version (21K):
[in a new window]
Fig. 5.
Determination of the kinetic parameters of
the GutR binding reaction to its target DNA. The biotinylated GutR
binding site was captured to the biosensor chip via immobilized
streptavidin on the chip surface. A, association rate
constant determination. Diamond, square,
triangle, and circle, the binding reaction
performed in the presence of GutR, GutR + glucitol, GutR + xylitol, and
GutR + glucose, respectively. The slope of each line reflects the value
of the association rate constant. B, dissociation rate
constant determination. Diamond, circle, and
triangle, binding reactions performed with GutR, GutR + xylitol, and GutR + glucose, respectively. The slope of each plot
reflects the value of the dissociation rate constant.
Rate constants for the interaction between GutR and its target sequence
determined by BIAcoreX biosensor
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
78 to
50 of the gut operon. This site is AT-rich. Both
the in vitro and in vivo deletion studies
indicate that there is only a single GutR binding site in this system.
Because the entire B. subtilis genome has been sequenced
(17), a pattern search of the SubtiList data base (18) for this
29-nucleotide GutR binding sequence was performed. With an allowance
even up to a seven-nucleotide mismatch in this 29-nucleotide region
during the search, the GutR binding site located upstream of the
gut operon is the only sequence that can be found.
10 M)
even in the absence of glucitol. If this is the case, what is the
physiological role of glucitol? With an off-rate of 1.7 × 10
3 s
1 determined at 25 °C, the
half-life (t1/2) of the GutR-DNA complex in the
absence of glucitol is estimated to be 6.8 min (Table I). Considering
that B. subtilis as a soil bacterium and that the
nutrients present in the soil are not always at high levels, the
generation time for cell division can be relatively long. By
cultivating B. subtilis in a defined medium (SP1) (21) at
25 °C, the generation time is determined to be 380 min (6.3 h).
Therefore, a half-life of 6.8 min of the GutR-DNA complex would not be
considered to be significant. In contrast, the BIAcoreX biosensor can
measure the off-rate as low as 1 × 10
5
s
1, and this translates to a half-life
(t1/2) of the complex to 19 h. In the presence
of glucitol, the dissociation rate constant for the GutR-DNA complex
should be less than 1 × 10
5 s
1
because it was beyond the limit that could be measured by the BIAcoreX
biosensor. Therefore, the GutR-DNA complex in the presence of glucitol
had the off-rate decreased by 100-fold or more and had the ability to
bind to its binding site without dissociation before the cell
divided. This tight binding raises an important question. Why
would GutR remain bound to its target site? This tight binding is
hypothesized here to allow GutR to be positioned very close to the
promoter as one of the necessary steps in a multistep process to
mediate the transcription activation process. Our study indicates that
the next step in the activation process is the binding of ATP to
GutR.2 Under this
condition, GutR has positioned itself at the proper location to
wait for the next step (ATP binding) to occur. Therefore, one of the
functions of glucitol as the inducer is to induce GutR to have a tight
binding to its target site so that subsequent reactions can occur and
lead to the activation of the gut operon.
70 family rather than the
E
54 family), and their binding sites are
located slightly upstream of the
35 region of the promoters. In the
absence of inducer, both MalT (26) and GutR exist as monomers in
solution. However, the GutR system has its unique aspects that are
different from the MalT system. MalT can bind to its binding sites only
in the presence of both the inducer (maltotriose) and ATP (27, 28). This is not the case for the GutR system. Multiple MalT binding sites
are located upstream of the mal promoters (29, 30), whereas
only a single GutR binding site is in the gut operon. Furthermore, the putative DNA binding site in GutR is located at the
N-terminal region rather than at the C-terminal region as observed for
MalT (29). All these differences indicate that GutR may mediate its
transcription activation by a mechanism that is different from other
known systems, and the elucidation of this mechanism would provide
insights to understand this transcription activation system.
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FOOTNOTES |
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* This project is supported by a research grant from the Natural Science and Engineering Research Council of Canada (to S. L. W.).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.
Supported in part by a research assistantship from the Department
of Biological Sciences at the University of Calgary. Current address:
University of Guelph, Dept. of Microbiology, Guelph, Ontario N1G 2W1, Canada.
§ To whom correspondence should be addressed. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong@ucalgary.ca.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M009864200
2 K. K. H. Poon and S. -L. Wong, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Gut, glucitol utilization.
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REFERENCES |
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