From the Departments of Biochemistry, the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
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ABSTRACT |
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D-Mannitol is taken up by
Bacillus stearothermophilus and phosphorylated via a
phosphoenolpyruvate-dependent phosphotransferase system
(PTS). The genes involved in the mannitol uptake were recently cloned
and sequenced. One of the genes codes for a putative transcriptional regulator, MtlR. The presence of a DNA binding helix-turn-helix motif
and two antiterminator-like PTS regulation domains, suggest that MtlR
is a DNA-binding protein, the activity of which can be regulated by
phosphorylation by components of the PTS. To demonstrate DNA binding of
MtlR to a region upstream of the mannitol promoter, by DNA
footprinting, MtlR was overproduced and purified. EI, HPr, IIAmtl, and IICBmtl of B. stearothermophilus were purified and used to demonstrate that
MtlR can be phosphorylated and regulated by HPr and
IICBmtl, in vitro. Phosphorylation of MtlR by
HPr increases the affinity of MtlR for its binding site, whereas
phosphorylation by IICBmtl results in a reduction of this
affinity. The differential effect of phosphorylation, by two different
proteins, on the DNA binding properties of a bacterial transcriptional
regulator has not, to our knowledge, been described before. Regulation
of MtlR by two components of the PTS is an example of an elegant
control system sensing both the presence of mannitol and the need to
utilize this substrate.
Many bacteria transport D-mannitol and other
carbohydrates via a phosphoenolpyruvate-dependent
phosphotransferase system
(PTS)1 (1-3). Two general
cytoplasmic proteins, EI and HPr, are responsible for the transfer of
the phosphoryl group from PEP to different sugar-specific PTS proteins.
Each sugar-specific system consists of three parts: IIA, IIB, and IIC.
IIC is the transmembrane transporter, responsible for the transfer of
the sugar across the cytoplasmic membrane. The transported sugar is
phosphorylated by IIB while it is still bound to IIC. IIA is
responsible for the transfer of the phosphoryl group from HPr to IIB.
Different fusions between IIA, IIB, and IIC occur naturally in the PTS.
For example, in the mannitol uptake system of Escherichia
coli, these proteins are covalently linked as one polypeptide
chain, IICBAmtl, whereas in B. stearothermophilus, the same system consists of a soluble
IIAmtl and a membrane-bound IICBmtl.
Recently, the entire mannitol operon of Bacillus
stearothermophilus was cloned (4). Four open reading frames,
mtlA, mtlR, mtlF, and mtlD,
were identified within the operon, coding for the mannitol transporter
(IICBmtl), a putative transcriptional regulator (MtlR),
enzyme IIAmtl, and the mannitol-1-phosphate dehydrogenase
(MPDH), respectively (Fig. 1). The
mannitol transporter IICBmtl was expressed, and its
involvement in the uptake of mannitol by B. stearothermophilus was confirmed (4). The sequence of the
mtlR gene resembles that of transcriptional regulators, such as antiterminators and repressors, and is, therefore, expected to be
involved in the regulation of the mannitol operon. In this paper, we
report the functional expression and isolation of the B. stearothermophilus EI, HPr, IIAmtl, and MtlR and the
analysis of the mtlR gene product, MtlR, that reveals its
function as a transcriptional regulator, involved in the regulation of
the mannitol operon.
Restriction enzymes, Taq-DNApolymerase, nucleotides,
oligonucleotide-kinase, pyruvate-kinase, IPTG,
5-bromo-4-chloro-3-indolyl- General Methods--
DNA was isolated from agarose gels using
the Quiagen gel extraction kit. Protein concentrations were determined
according to Bradford (9). General DNA manipulations were performed as described in Sambrook et al. (10). Sequence data base
searches were performed using the program BLAST at the National Center for Biotechnology Information (11).
Construction of pETMtlR-his--
Overexpression of the
mtlR gene was established with the T7 Expression and Isolation of MtlR-his--
A preculture of
BL21-DE3 with pETMtlr-his was grown overnight at 30 °C and was
diluted 100-fold in 5-liter flasks with 0.5 liters of LB medium (10 g
of trypton, 5 g of yeast extract, and 10 g of NaCl per liter)
The cultures were grown at 30 °C with vigorous shaking at 300 rpm.
At A660 = 0.6, the culture was induced with 0.8 mM IPTG and grown for 90 min, after which the cells were collected by centrifugation (3000 × g for 10 min at
4 °C). The pellet was washed in 0.5 liter of 25 mM Tris,
pH 8.0, and 0.2 M KCl and resuspended in 20 ml of 25 mM Tris, pH 8.0, 1 M NaCl, 3 mM
Expression and Purification of EI and HPr of B. stearothermophilus--
EI and HPr of B. stearothermophilus
were expressed from the plasmid pLOI1801 in the EI, HPr deletion mutant
E. coli ZSC-112 as described by Lai and Ingram (12). A
culture of ZSC-112 containing pLOI1801 was grown overnight at 37 °C,
after which the cells were collected by centrifugation. The pellet was
washed in 10 mM Tris, pH 7.5, and 1 mM DTT and
resuspended in 10 mM Tris, pH 7.5, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM
MgCl2, and 0.2 mg/ml DNase and RNase. The cells were
ruptured with a French press at 10,000 psi, and 2.5 mM EDTA
was added. Whole cells and precipitates were removed by centrifugation
at 20,000 × g. The supernatant was dialyzed against 10 mM Tris-HCl, pH 7.5, and 2 mM DTT for 16 h
and applied to a 40-ml Q-Sepharose column. The column was washed with 2 column volumes of Bis-Tris, pH 7.0, 1 mM DTT, and the
proteins were eluted with a 400-ml linear gradient from 1 to 600 mM NaCl in buffer. HPr eluted in the wash step and was purified to homogeneity by gel filtration on a G75 column. The EI
eluted at 350 mM NaCl and was directly used in the further experiments, although some impurities were still present.
Expression and Purification of IIAmtl of B. stearothermophilus--
IIAmtl was expressed as a fusion
product with glutathione S-transferase (GST) using the
plasmid pGEX-2T. To enable the insertion of the mtlF gene
into pET15-b, a BglII and an EcoRI restriction site were created, by PCR, at the start and downstream of the mtlF gene, respectively. The PCR was performed as described
for the construction of pETMtlR-his using the same template but with two other mutagenic primers (N terminus, 5'-GTG AAT GAC AGA TCT ATG CCA ATT-3'; C terminus, 5'-GGA ATG AAT TCC TAA
ACA TGC-3'). PCR fragments of 478 bp were isolated from the gel and
ligated into the EcoRV site of pSK
For the expression of the GST-IIAmtl fusion product,
E. coli Jm101 with pGEXIIA was grown to
A660 0.85, induced with 0.5 mM IPTG, and grown overnight. 4 g of cells were collected by centrifugation at 6000 × g, washed in 50 mM NaPi, pH 7.5, resuspended in 20 ml of 50 mM NaPi, pH 7.5, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl2, and 0.2 mg/ml DNase and RNase. After
disruption with a French press, whole cells and precipitates were
removed by centrifugation at 20,000 × g followed by the addition
of 2.5 mM EDTA, 150 mM NaCl, and 2.4 g
(dry weight) of glutathione agarose equilibrated in 50 mM
Tris, pH 7.5, and 150 mM NaCl. The mixture was incubated with continuous agitation for 1 h at room temperature and
collected in a column. Unbound protein was removed by washing with 10 column volumes of equilibration buffer. The column material was
resuspended in 50 ml of equilibration buffer containing 0.3 mM CaCl2 and 150 mg of thrombin and incubated
overnight at room temperature. Soluble IIAmtl, cleaved from
the bound GST, was collected by filtration. Thrombin, also present in
the filtrate, was precipitated by incubating the sample for 30 min at
60 °C. Pure IIAmtl was then obtained by centrifugation.
Phosphorylation of MtlR--
[32P]PEP was
synthesized, following the method of Roossien et al. (13).
B. stearothermophilus IICBmtl-containing
membrane vesicles and purified IICBmtl were created as
described by Henstra et al. (4). MtlR, enzymes, and vesicles
were diluted in phosphorylation buffer (25 mM Tris-HCl, pH
7.5, 0.5 mM MgCl2, 1 mM NaF, and 5 mM DTT). The reaction was started by adding
[32P]PEP and stopped, after incubation at 30 °C, with
1/2 volume denaturation buffer. Following separation of the
proteins on a 15% SDS-polyacrylamide gel by electrophoresis,
phosphorylation of proteins was visualized on a Molecular Dynamics
PhosphorImager 425. The autoradiogram was analyzed using the Image
Quant program.
DNA Footprint--
A single-end 32P-labeled DNA
probe of the mannitol promoter region was synthesized in a PCR in which
one of the primers was labeled. 28.5 pmol of the forward primer sah1
(5'-GGC AGG TGA ATT GTT AAA G-3') priming at position
The binding of MtlR to the target DNA was achieved by mixing the sample
with 25 µl of binding buffer (20 mM Tris-HCl, 80 mM KCl, 25% glycerol, 8 mM MgCl2,
and 2 mM DTT, pH 8.0) and triple distilled water to a final
volume of 46 µl. Each reaction was started by adding 4 µl of 20-40
kcpm DNA probe (~50 fmol of DNA) to the mixture, followed by an
incubation of 15 min at 30 °C. The DNA was digested by adding 50 µl of 10 mM Tris, pH 8.0, 10 mM
MgCl2, 5 mM CaCl2, 0.03 units/µl
of RQ1-DNaseI and incubated for 2 min at room temperature. The
digestion was stopped with 90 µl of 0.6 M NaAc, 30 mM EDTA, 0.5% SDS, and 30 µg/µl yeast tRNA. The digest
was then purified by a chloroform phenol extraction and precipitated
with 2.5 volumes of EtOH at Primer Extension--
Samples of total RNA were isolated from
E. coli or B. stearothermophilus grown on minimal
medium supplemented with 0.5% glucose, mannitol, or mannitol plus
glucose. Exponentially growing cells were harvested at an
A660 of 0.5 and RNA was extracted as described by Ausubel et al. (15). For the primer extension reaction,
10 pmol of primer sah2 (3'-GACCGAAAGCACATTTCTAAGTCGCAAAAC-5'),
at position +106 to +82, was labeled with 30 µCi of
[ Phosphoenolpyruvate-dependent Mannitol
Phosphorylation Assay--
PEP-dependent mannitol
phosphorylation activity was measured as described by Robillard and
Blaauw (16). The assays were performed in a 100-µl assay mixture
containing 25 mM Tris, pH 7.5, 5 mM
MgCl2, 0.25% decyl-polyethylene glycol, 5 mM
DTT, 5 mM PEP, 33 µM
[14C]mannitol and 0.04 mg/ml EI, 18 µM HPr
and 24 µM IIAmtl, all from B. stearothermophilus.
Mannitol-1-phosphate Dehydrogenase Assay--
MPDH activity was
measured as described by Novotny et al. (17). The sample was
incubated 3 min in 70 mM Tris-HCl (pH 9) and 1 mM NAD+ at 20 °C. Conversion of
NAD+ to NADH was followed for 7 min at 340 nm, after
starting the reaction with 4 mM mannitol 1-phosphate.
Sequence of the mtlR Gene--
The mtlR gene was cloned
as part of the mannitol operon and sequenced as described by Henstra
et al. (4). The gene of 2091 bp codes for a protein of 697 amino acids with a calculated mass of 79 kDa. In the protein sequence,
conserved areas are found that show resemblance to domains of two
different types of transcriptional regulators (Fig.
2). First, a helix-turn-helix motif (Fig.
2, black box) was identified at the N terminus using the
method described by Dodd and Egan (18), which shows similarity with the
HTH motifs of members of the DeoR family of transcriptional regulators,
such as the fucose (19), glucitol (20), and deoxyribonucleotide (21)
repressor of E. coli and the lactose regulator of
Lactococcus lactis (22) and of Streptococcus
mutans (23). Sequence similarity to the rest of these regulators
is low (10-18% overall identity), with the exception of a region
that, in case of the DeoR and the LacR regulator, is involved in
binding the inducer. These inducer binding sites are the targets of
phosphorylated substrates, such as galactose 6-phosphate in the case of
LacR of L. lactis (24). Second, the central part of the
protein contains sequences similar to the C terminus of antiterminators
of the BglG family, such as BglG (25) of E. coli and SacT
(26), SacY (27), GlcT (28), and LicT (29) of Bacillus
subtilis. Two homologous PTS regulation domains, PRD-I and PRD-II
(Fig. 2, gray boxes), are located in this part of the
protein. They appear to be involved in the regulation of the activity
of the antiterminator via phosphorylation by PTS components (30-34).
All four putative phosphorylation sites, histidines 235 and 294 in
PRD-I and histidines 348 and 405 in PRD-II, and their surroundings are
conserved in most of the antiterminators and MtlR, suggesting that MtlR
could also be controlled by phosphorylation by EI and HPr and the
mannitol-specific components IIAmtl and
IICBmtl. Combinations of a DNA binding motif with an
antiterminator-like motif have been found before for the levanase
regulator LevR (35) and the licheanan regulator LicR (36) of B. subtilis. The homology between MtlR and these proteins is limited
to the regions involved in DNA binding and in phosphorylation by the
PTS. Recently, the gene ydaA, discovered by the B. subtilis genome sequence project, was submitted to GenBankTM
(accession number AB001488). The product of this gene has the same
topology as the B. stearothermophilus MtlR and a 39%
overall sequence identity; it is the protein most similar to MtlR found
in the sequence data bases. The gene is not located near the mannitol
operon of B. subtilis, but it could be involved in the
regulation of this operon. Two other Gram-positive mannitol operons
possess parts of genes that show similarity to MtlR. First, an open
reading frame is found within the S. mutans mannitol operon
that codes for a sequence similar to the C terminus of MtlR (37).
Second, Staphylococcus carnosus possesses an open reading
frame of 150 amino acids downstream of mtlA that contains a
HTH motif with 52% identity compared with the HTH in MtlR.
The Mannitol Promoter of B. stearothermophilus--
The high
homology between the above-mentioned proteins and the B. stearothermophilus MtlR suggest that the mannitol operons of these
microorganisms can be regulated in a comparable way. A conserved
sequence of 85 bases was revealed in the putative promoter regions
upstream of the mtlA genes of B. stearothermophilus, B. subtilis, and S. carnosus (Fig. 3). The identity
between these regions in B. stearothermophilus and B. subtilis or B. stearothermophilus and S. carnosus was 67 and 47%, respectively. A putative CRE sequence (38), involved in catabolite repression, is found in all three promoter
regions. Deviations from the consensus CRE sequence (39) are found at
one position for the B. stearothermophilus and B. subtilis and at three positions for the S. carnosus CRE
box. A conserved putative Expression and Purification of MtlR--
The MtlR gene was cloned
in the T7 expression plasmid pET-15b (see under "Materials and
Methods"), creating a fusion of a His tag and a thrombin cleavage
site at the N terminus of MtlR. The protein was successfully expressed
from the plasmid pETMtlR-his in E. coli BL21-DE3 (Fig.
5, lane 2). After disruption
of the cells, part of the protein was found in an insoluble form and was removed by centrifugation (lane 3). The ratio of soluble
to insoluble protein could be improved by lowering the temperature and
increasing agitation during growth. Also, the addition of 1 M NaCl to the disruption buffer decreased the amount of
precipitated protein in the crude extract. Most of the soluble
His-tagged MtlR (Fig. 5, lane 4) was bound to the NTA
Ni-agarose. After the column material was washed, the protein was
eluted with a purity greater than 95% (Fig. 5, lane 5). The
mass was determined to be 81,590 Da by matrix-assisted laser desorption
ionization-time of flight mass spectroscopy. The calculated mass of the
fusion product, 81,545 Da, was within the experimental error of the
determined mass (data not shown). Analysis of the purified protein by
Western blotting using His tag-directed antibodies (data not shown)
revealed that the remaining impurities visible on Coomassie-stained
SDS-PAGE are N-terminal degradation products of the MtlR protein.
Serious protein degradation was observed during the early
purifications. This problem was solved by harvesting cells before they
enter the stationary growth phase.
Purification of EI, HPr, and IIAmtl of B. stearothermophilus--
B. stearothermophilus EI and HPr,
expressed simultaneously in the EI and HPr deficient E. coli
ZSC112, were separated from each other and from other proteins on a
Q-Sepharose ion exchange column. The partly purified EI fraction (Fig.
5, lane 6) was not purified to homogeneity but used directly
in subsequent experiments. HPr was purified to homogeneity by gel
filtration (Fig. 5, lane 7).
The GST-IIAmtl fusion product could be easily separated
from the other E. coli proteins by binding to glutathione
agarose. IIAmtl was cleaved from the column bound GST by
digestion with thrombin. The removal of thrombin by precipitation at
60 °C did not affect the activity of the purified
IIAmtl. This procedure yielded a homogenous
IIAmtl preparation (Fig. 5, lane 8). The N
terminus of IIAmtl was sequenced, which confirmed the
sequence GSMPILKKE, which was expected after digestion of the fusion
product by thrombin. The masses of HPr and IIAmtl were
determined by matrix-assisted laser desorption ionization-time of
flight mass spectroscopy to be 9032.5 and 15,958 Da, respectively. They
correspond well to the theoretical masses of HPr without the first
methionine (9035 Da) and IIAmtl with the expected
replacement of the first methionine by a glycine (15, 950 Da).
Phosphorylation of MtlR by HPr and
IICBmtl--
Purified MtlR was phosphorylated in the
presence of partially purified components of the mannitol PTS, with
exception of IICBmtl, which was added as inside-out
E. coli LGS322 vesicles expressing B. stearothermophilus IICBmtl. PEP-dependent
protein phosphorylation was performed with [32P]PEP and
was visualized with a Phosphorimager after separation by SDS-PAGE (Fig.
6). The phosphorylation characteristics
of MtlR were investigated by adding, one by one, the successive
components of the PTS in the presence (Fig. 6A, lanes 5-8)
and absence (lanes 1-4) of MtlR. Phosphorylation of the
regulator was most obvious if all of the components of the B. stearothermophilus mannitol PTS, EI, HPr, IIAmtl, and
IICBmtl were added (lane 8). Phosphorylation of
MtlR by EI and HPr alone was also observed, although the amount
phosphorylated MtlR was much lower than was found by phosphorylation
via IICB. Phosphorylation by HPr became more evident when the HPr
concentration was raised from 0.18 to 3.6 µM (Fig.
6B, lanes 1-4) or when the reaction time was increased from
15 to 100 min (Fig. 6B, lane 5). Addition of EI alone or
IIAmtl to the EI/HPr mixture did not affect the
phosphorylation level of MtlR. We therefore concluded that MtlR can be
phosphorylated by both HPr and IICBmtl. The presence of the
substrate mannitol in the reaction mixture during the entire incubation
period reduced the amount of phosphorylated MtlR (Fig. 6A, lane
9). Addition of mannitol in only the last 1 min of the incubation
period also resulted in a reduced of level of phosphorylated MtlR (Fig.
6A, lane 10). This suggests that the phosphorylation of MtlR
by IICBmtl is reversible.
It is obvious from a comparison of Fig. 6A and Fig.
6B that a much higher concentration of HPr versus
IICBmtl is necessary to get comparable levels of MtlR
phosphorylation. In order to quantify this difference, the rate of MtlR
phosphorylation was determined by following the time dependence of the
phosphorylation levels of the MtlR band (see Fig.
7, A and B). The
HPr and IICBmtl dependence of the MtlR phosphorylation rate
was calculated from the linear portion of the plot to be 0.021 and 31 pmol min
To prove reversible phosphorylation of MtlR by IICBmtl,
MtlR was phosphorylated under conditions where the protein is mainly phosphorylated by IICBmtl and purified on Ni-NTA agarose.
32P-MtlR was mixed IICBmtl and incubated in the
presence and absence of mannitol. The decrease in the amount of
phosphorylated MtlR was followed against time with SDS-PAGE, quantified
using the phosphorimager, and plotted Fig. 7C. The half-time
of the phosphorylated MtlR is 15 and 119 min in the presence and
absence of mannitol, respectively. This demonstrates that MtlR can be
dephosphorylated by IICBmtl and mannitol.
Binding MtlR to the Mannitol Promoter--
Footprint experiments
were performed to determine whether MtlR is a DNA-binding protein and
to locate the possible binding site. Based upon the homology between
the promoters of B. subtilis, S. carnosus, and
B. stearothermophilus, we expected that the region between
The Effect of Phosphorylation on the Binding of MtlR to the
Mannitol Promoter--
Because phosphorylation of MtlR by components
of the PTS could be involved in the regulation of the activity of the
regulator, we studied the influence of phosphorylation of MtlR on its
affinity for its binding site. To investigate this, we performed DNA
footprint experiments under various phosphorylation conditions. The
difference in kinetics of the phosphorylation of MtlR by HPr and IICB,
as demonstrated in Fig. 7, were used to obtain MtlR phosphorylated mainly by HPr, IICBmtl, or HPr and IICBmtl.
MtlR-dependent DNA footprints were made with MtlR
phosphorylated by different compounds of the PTS and are presented in
Fig. 9A. The protection level
of footprint FP1 was measured and is plotted against the MtlR
concentration in Fig. 9B. The other protected areas, FP2 to
FP5, showed MtlR concentration dependences similar to that of FP1 and
are not included in Fig. 9B. Differences in the
concentration dependence of the protection were observed under different phosphorylation conditions. Reaction conditions that favor
dephosphorylation of MtlR were created by adding all components of the
PTS in the presence of mannitol but in the absence of PEP (reaction V).
Under these conditions, 50% protection was observed at a MtlR
concentration of 92 nM. When the protein was incubated with
PEP, EI, and high concentrations of HPr, the affinity of MtlR for its
binding site increased by a factor of 4, to 22 nM (reaction
I). This indicates that phosphorylation of MtlR by HPr has a positive
effect on the DNA binding properties of MtlR. Phosphorylation of MtlR
by IICB, using PEP, EI, IIA, IICBmtl, and low
concentrations of HPr, resulted in a 14-fold decrease in the affinity,
to 1300 nM (reaction II), compared with the
nonphosphorylated protein in reaction V. Clearly, phosphorylation of
MtlR by IICBmtl has a negative effect on the DNA binding
properties of MtlR. Phosphorylation of MtlR by both HPr and
IICBmtl, using high concentrations HPr, results in a slight
increase in binding, to 720 nM (reaction III), compared
with MtlR phosphorylated only by IICBmtl. The strongest
binding of MtlR to the DNA is observed under conditions where MtlR is
phosphorylated by PEP, EI, and HPr and simultaneously dephosphorylated
by IICBmtl and mannitol (reaction IV). Under these
conditions, an affinity of 3.4 nM was measured that was 27 times higher than the affinity of the nonphosphorylated MtlR in
reaction V and 382 times higher than the affinity of MtlR
phosphorylated by IICBmtl in reaction II. It is important
to emphasize that the MtlR used for these experiments had to be
dephosphorylated first. Because of the rapidity of the Ni-NTA isolation
procedure of the His-tagged MtlR and the use of buffers with a pH of 8, the protein, as isolated, is still phosphorylated. Consequently, the
effects described above were only observed if the protein was incubated
at pH 6.5, to accelerate hydrolysis of the phosphohistidines present in
the isolated MtlR. The change in the level of MtlR phosphorylation by
this treatment was estimated by adding IICBmtl and
[3H]mannitol and monitoring the formation of
[3H]mannitol 1-phosphate.
The Activity of the Mannitol Promoter in Vivo--
The intensity
of the signal obtained by the primer extension experiment in Fig. 4 is
dependent on the growth substrate used. Glucose lowers the intensity of
the signal, indicating catabolite repression of the mannitol operon by
glucose. The presence of a mannitol regulator and a CRE site already
suggest that expression of this operon is induced by mannitol and
repressed by glucose. To confirm this suggestion, B. stearothermophilus was grown on mannitol, glucose, and mannitol
plus glucose. Expression levels of the first and the last gene of the
mannitol operon were determined by measuring the IICBmtl
and the MPDH activity, respectively (Table
I). The IICBmtl activity was
measured using the purified B. stearothermophilus EI, HPr,
and IIAmtl. When grown in the presence of glucose as the
only energy and carbon source, the activity of IICBmtl and
MPDH are low. The activity of both enzymes increases if the cells are
grown on mannitol instead of glucose by factors of 24 and 14, respectively. Expression levels found with growth on mannitol can be
repressed by the addition of glucose to the growth medium. However, the
extent of repression for both enzymes is not equal. The
IICBmtl specific activity decreases by a factor of 4.6, whereas the MPDH specific activity decreases by a factor of 1.9 upon
the addition of glucose to the medium. Involvement of a second
promoter, located between mtlA and mtlD, could be
an explanation for this observation. A possible position for such a
second promoter could be the 284-bp gap between the mtlA and
mtlR genes. Two putative promoters are assigned in this
region by the method of Reese et al. (41).
Analysis of the promoter region of the B. stearothermophilus operon revealed a
MtlR can be phosphorylated both by HPr and IICBmtl, as was
demonstrated in vitro, using purified B. stearothermophilus EI, HPr, and IIAmtl and membrane
vesicles containing IICBmtl. The most probable targets for
phosphorylation are the phosphorylation domains PRD-I and PRD-II, which
are conserved in antiterminators and in LicR and LevR. The N-terminal
PRD-I domain in LevR is phosphorylated by HPr, whereas the C-terminal
PRD-II domain is phosphorylated by LevE, a IIB-like protein (42).
Phosphorylation by HPr was also demonstrated in vitro for
the antiterminators SacY and SacT (34, 43). Phosphorylation of SacY
(44) and BglG (31) by the sugar-specific PTS proteins, SacX and BglF,
respectively, has been observed. The phosphocysteine in the B-domain of
BglF is the phosphoryl donor both for the carbohydrate and for BglG (45). A similar competition between mannitol and MtlR takes place in
the mannitol system, because the addition of mannitol results in the
dephosphorylation of MtlR by IICBmtl.
Studies on several antiterminators, as well as LevR and LicR, revealed
that phosphorylation of these proteins enhance their regulating
activity (recently reviewed by Stülke (30)). Transcriptional activation by LevR, SacT, and LicT is dependent on or is stimulated by
the presence of EI and HPr. Phosphorylation of these proteins by HPr is
most likely the activating signal. Negative control by sugar-specific
components of the PTS was observed in vivo in the case of
LevR, LicT, SacY, SacT, and BglG (32, 33, 46-49), probably caused by
the phosphorylation of the regulator by one of the sugar-specific PTS
components. The effect of phosphorylation on the activity of
antiterminators and LevR has been determined in vivo, using
mutants with defects in the PTS phosphorylating proteins or mutant
regulators in which the putative phosphorylation sites have been
replaced. Only in the case of BglG, and now also MtlR, has a direct
effect of phosphorylation been observed. The phosphorylation of BglG by
the Phosphorylation of MtlR changes the affinity of MtlR for its binding
site. A maximum 382-fold difference in affinity was observed under
different phosphorylation conditions. Phosphorylation of MtlR by HPr
results in an increase of the affinity of the MtlR for the DNA, whereas
phosphorylation by IICBmtl results in a decrease. The
negative effect of the phosphorylation of MtlR by IICBmtl
dominates the positive effect of the phosphorylation by HPr, because
the difference in affinity between MtlR phosphorylated by
IICBmtl alone or IICBmtl plus HPr is minor. If
MtlR is a transcriptional activator, which is regulated in a way
comparable to LevR, phosphorylation of one of the PRD domains by HPr
should result in an increased binding to the DNA, whereas
phosphorylation of the other PRD domain by IICBmtl should
result in a decreased affinity. The highest affinity of MtlR for the
DNA was observed when the protein was phosphorylated by PEP, EI, and
HPr and simultaneously dephosphorylated by IICBmtl and
mannitol. This observation could be explained by the observation that
HPr can phosphorylate at least three histidines of SacY, one on PRD-I
and two on PRD-II (34). If HPr could phosphorylate a second site in
MtlR, the same site phosphorylated by IICBmtl, it could
reduce the activation obtained by phosphorylation of the activation
site. In the presence of IICBmtl and mannitol, this second
site will be dephosphorylated, allowing maximal stimulation by HPr.
Mutagenesis studies2 indicate
that both His235 and His294 in PRD-I and
His348 and His405 in PRD-II are necessary for
IICBmtl- and HPr-dependent phosphorylation,
respectively. Based on the same studies, indications for a third
phosphorylation domain have been found, that probably can be
phosphorylated by IICBmtl resulting in a decreased affinity
of MtlR for the DNA. Phosphorylation of this site by HPr could be an
explanation for the reduced activation by HPr in the absence of
IICBmtl and mannitol.
The observations described above fit well in a regulation model already
proposed for LevR by Stülke et al. (30, 46) and presented in Fig. 10. Regulation of the
mannitol operon would occur by two different mechanisms, (i) catabolite
repression, a MtlR-independent mechanism, and (ii) transcriptional
activation, a MtlR-dependent mechanism. Catabolite
repression is mediated by the presence of a CRE box. High levels of
fructose 1,6-bisphosphate, a signal of high internal energy levels,
would stimulate a kinase to phosphorylate HPr on a serine. The binding
of the P-ser-HPr:CcpA complex to the CRE box would result in the
inactivation of the mannitol promoter. Transcriptional regulation is
suggested by the presence of MtlR. Binding of the regulator to a site
upstream of the mannitol promoter could result in the activation of the
expression of the mannitol operon. The affinity of MtlR for this site
is regulated by phosphorylation of MtlR by the general PTS component
HPr and the mannitol-specific component IICBmtl.
IICBmtl acts not only as the mannitol transporter but also
as a mannitol sensor. When mannitol is absent, the concentration
phosphorylated IICBmtl will be high, resulting in the
phosphorylation of MtlR at the IICBmtl-specific site. This
causes the inactivation of MtlR as a transcriptional activator by
preventing binding to the mannitol operon. However, if mannitol is
present, IICBmtl will transfer the phosphate group mainly
to mannitol and not to MtlR, preventing inactivation of MtlR.
Phosphorylation by HPr can be considered as a CcpA-independent
mechanism of catabolite repression. High uptake rates of PTS
substrates, such as glucose and fructose, but also mannitol, result in
decreased concentrations of P-his-HPr. MtlR will not be phosphorylated
by HPr. However, if the PTS-dependent uptake activity is
low, most of the HPr will be present as P-his-HPr, phosphorylate MtlR
on its activation domain, and stimulate expression of the mannitol
operon by binding upstream of the mannitol promoter. Whether
stimulation by P-his-HPr takes place depends on the presence of
mannitol, because the negative effect of phosphorylation by
IICBmtl dominates the positive effect of phosphorylation by
HPr on the binding of MtlR to the promoter region. The suggestion that
the HPr-dependent stimulation by phosphorylation of MtlR on
one site can be reduced by phosphorylation of a second site by HPr, a
site that can also be dephosphorylated by IICBmtl, fits the
proposed model. In this case, initial phosphorylation of MtlR by HPr
will result in a reduced activation of the expression of the mannitol
operon. Only when mannitol is present will the expressed
IICBmtl dephosphorylate this second site and allow full
stimulation by HPr.
INTRODUCTION
Top
Abstract
Introduction
References
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Fig. 1.
Schematic overview of the mannitol operon and
subclones used to create the expression vector pETMtlR-his and the
subclones involved in the DNA footprint, promoter activity, and primer
extension experiments. The genes of the mannitol operon, the
mannitol and T7 virus 10 promoter, and the His tag are indicated by
open arrows, open and filled
triangles, and open box, respectively. Restriction
enzymes BamHI, ClaI, EcoRI,
HindIII, SalI, and XbaI are indicated
by B, C, E, H, S, and X, respectively.
MATERIALS AND METHODS
-D-galactopyranoside, and the
first strand cDNA synthesis kit for reverse transcription-PCR (Avian Myeloblastosis Virus) were purchased from Boehringer Mannheim. [
-32P]ATP (3000Ci/mmol) and
[14C]mannitol (50-62 mCi/mmol) were obtained from
Amersham, Ni-NTA agarose from Qiagen, and PET15b from Novagen.
RNase-free RQ1-DNase I was obtained from the Promega core footprinting
system. Thrombin, with a specific activity of 8165 units/mg of protein,
was from ICN Biomedicals Inc. Primers were synthesized by Eurosequence. PEP and yeast tRNA were purchased from Sigma. The vectors
pSK
, pET15-B, and pGEX-2T were obtained from Stratagen,
Novagen, and Amersham Pharmacia Biotech, respectively. All DNA
manipulations and the expression of B. stearothermophilus
IIAmtl were performed in E. coli JM101 (5). The
MtlR gene was overexpressed in E. coli Bl21(DE3) (6). The
B. stearothermophilus mannitol transporter,
IICBmtl, was expressed in the mannitol deletion E. coli strain LGS322 (7), as described by Henstra et al.
(4), and the B. stearothermophilus EI and HPr were expressed
in E. coli ZSC112 (8).
expression vector pET15-b, which enables a thrombin cleavable histidine-tag to be fused to the N terminus of the protein. To enable
the insertion of the mtlR gene into pET15-b, a
BamHI restriction site was created at the start and
downstream of the MtlR gene, with PCR using two mutagenic primers (N
terminus, 5'-GGG GCA ATG GAT CCA TCT GCA CGC-3'; C terminus,
5'-AGG AGT GAA TGA CAT GTG GAT CCC AAT TT-3'). The PCR was
performed in a total volume of 100 µl with 10 mM
Tris-HCl, pH 8.0, 5 mM MgCl2, 100 mM NaCl, 1 mM
-mercaptoethanol, 1 mM dNTPs, 1 µg of each primer, and 2.5 units of Pwo-DNA
polymerase. 100-500 pg of pSKEB5.5 was added as template DNA. This
plasmid contains a 5.5-kilobase pair DNA fragment of the mannitol
operon, including the entire mtlR gene (4). After 25 cycles
comprising 1 min of denaturation at 94 °C, 1 min of primer annealing
at 30 °C, and 2 min of extension at 72 °C, the PCR product was
separated by agarose gel electrophoresis. Fragments of 2116 bp were
isolated from the gel, cut with BamHI, and ligated into the
BamHI site of pSK
to create pSKMtlR (Fig. 1).
Positive clones were identified by blue/white screening on IPTG,
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside, ampicillin plates. The sequence of the insert of one of the positive clones was checked by sequencing. The MtlR gene in pSKMtlR was ligated
into the BamHI site of pET15b after digestion with
BamHI creating the expression vector pETMtlR-his.
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl2, and 0.2 mg/ml DNase and RNase per g
of cells. The cells were ruptured with a French press at 10,000 psi,
and 2 mM EDTA was added. Whole cells, cell debris,
and precipitate were removed by centrifugation at 3000 × g for 10 min; membranes were removed by centrifugation at
200,000 × g for 30 min. 1 ml of Ni-NTA resin per g of cells and 2 mM phenylmethylsulfonyl fluoride were added to the
supernatant, and the mixture was incubated with continuous agitation
for 30 min at 4 °C. The Ni-NTA resin was collected in a column and
subsequently washed with 10 column volumes of wash buffer (25 mM Tris-HCl, pH 8, 1 M NaCl, 3 mM
-mercaptoethanol, and 40 mM imidazole). The protein was
eluted by resuspension of the resin in 5-10 ml of wash buffer
containing 200 mM imidazole. After removal of the Ni-NTA
resin by filtration, 15 mM EDTA was added, and the
purified protein was dialyzed twice against 40 volumes of 25 mM Tris-HCl, pH 8, 0.2 M KCl, and 3 mM
-mercaptoethanol.
to create
pSKMtlF. The sequence of the insert of one of the positive clones was
checked by sequencing. The mtlF gene in pSKIIA was ligated
into the BamHI and EcoRI sites of pGEX-2T after
digestion with BglII and EcoRI creating the
expression vector pGEXIIA.
127 to
109
was labeled with 100 µCi of [
-32P]ATP (3000 Ci/mmol)
by T4-polynucleotide kinase as described by Boehringer Mannheim. The
labeled primer was purified by chloroform/phenol and chloroform
extractions followed by an ethanol precipitation. 19 pmol of the
labeled forward primer was built into a 473-bp probe by PCR in a
mixture containing 10 mM Tris, 1.5 mM
MgCl2, 50 mM KCl, 200 µM dNTPs,
2.5 units of Taq DNA polymerase, 57 pmol of universal
reverse primer (5'-ACAGGAAACAGCTATGACC-3') and 1 ng of template DNA.
The pSK-derived subclone pSKCH550, containing the area of the mannitol
promoter from ClaI (position
354) to HindIII
(position +212), was used as template DNA. After 30 cycles of 1 min of
denaturation at 94 °C, 1 min of annealing at 55 °C, and 1 min of
elongation at 72 °C, the 473-bp PCR product was separated by
electrophoresis on a 0.8% agarose gel and isolated from the gel with a
gel extraction kit from Quiex.
20 °C. Samples were separated on a 5%
sequencing gel. A reference sequence ladder was generated by PCR as
described by Krishnan et al. (14) with the same labeled
primer and template used for the synthesis of the probe.
-32P]ATP (3000 Ci/mmol) by T4-polynucleotide kinase
as described by Boehringer Mannheim. Each reaction was performed with
10 µg of total RNA and 500 kcpm of primer, according to the manual of the Boehringer Mannheim first strand cDNA synthesis kit for reverse transcription-PCR. The samples were analyzed on a sequencing gel together with a sequencing reaction performed with the same labeled primer.
RESULTS
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Fig. 2.
Schematic presentation of the topology of the
sequence of several transcriptional regulators similar to MtlR.
Shown are the fucose repressor of E. coli FucR (P11554); the
putative mannitol regulators MtlR (U18943) of B. stearothermophilus, YdaA (P96574) of B. subtilis, and
the N terminus of the MtlR of S. carnosus (P28008); the
transcriptional regulators LicR (previously called CelR) (P46321) and
LevR (P23914) of B. subtilis; and the antiterminators BglG
(P11989) and SacY (P15401) of E. coli and B. subtilis, respectively (All accession numbers are from Swiss.
Prot. except Vidgy3 which can be obtained at GenBank). The distinct
domains found in MtlR with similarities to the other listed proteins
are displayed as a black box for the DNA binding HTH motif,
a horizontally striped box for the putative inducer binding
sites, and two gray boxes representing the PTS regulation
domains PRD-I and PRD-II. The -factor binding domain, found only in
LevR, is indicated by the dotted box, and a
IIAmtl-like domain, found only in LicR, is indicated by the
vertically striped box. The positions of the conserved
histidines, probably involved in phosphorylation, are indicated by
H. Deviations of one of the conserved histidine in LevR and
BglG to an isoleucine and a aspartate are indicated by I and
N, respectively.
A-dependent
35
sequence was found 6-7 base pairs upstream of the CRE box for all
three organisms (40). The accompanying
10 region was located 17 bp
downstream of the
35 sequence, overlapping the CRE box in B. stearothermophilus and B. subtilis. In S. carnosus, no clear
10 sequence could be found at this position.
Prediction of the transcription start point, by the method of Reese
et al. (41), confirmed the B. stearothermophilus
and B. subtilis promoter but suggested a 4-bp shift upstream
for the S. carnosus promoter. The B. stearothermophilus transcription start was confirmed by primer
extension using RNA isolated from B. stearothermophilus grown on mannitol, mannitol plus glucose, and glucose. The result is
presented in Fig. 4 and shows that
transcription is started at an adenine 7 bp downstream from the
10
sequence.
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Fig. 3.
Sequence similarity between the mannitol
promoter region of B. subtilis, B. stearothermophilus, and S. carnosus and the
location of the footprints shown in Fig. 8. Identical bases
in the B. subtilis or S. carnosus and B. stearothermophilus promoter region are indicated by
asterisks. The predicted 10 and
35 regions are indicated
by gray boxes, and the CRE boxes matching the CRE consensus
sequence are indicated by lines. The predicted
transcriptional start sites are shown in boldface. The
position of the experimentally determined transcriptional start of the
B. stearothermophilus mannitol promoter is indicated by the
arrow. The regions protected by MtlR against DNase I
digestion are shown as black boxes and marked with
FP1-FP5.
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Fig. 4.
Determination of the transcriptional start of
the mannitol operon. Primer extensions products were obtained from
RNA isolated from B. stearothermophilus grown on mannitol
(lane 1), mannitol plus glucose (lane 2), and
glucose (lane 3) as described under "Materials and
Methods." The sequence ladder was obtained with the same primer used
for the primer extension reaction. On the right is the
position of the 10 sequence, and the start site, +1, is
indicated by an arrow.
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Fig. 5.
Overview of the expression and isolation of
B. stearothermophilus MtlR and the purified B. stearothermophilus PTS proteins EI, HPr,
IIAmtl, and IICBmtl
visualized by SDS-PAGE. Lanes 1 and
10, protein standards; lane 2, crude cell extract
of BL21-DE3 with pETMtlR-his; lane 3, insoluble MtlR and
protein removed by centrifugation; lane 4, the soluble
fraction; lanes 5-9, the purified MtlR, EI, HPr,
IIAmtl, and IICBmtl, respectively. Samples were
analyzed on a 15% SDS-polyacrylaminde gel, and the gel was stained
with Coomassie Blue.
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Fig. 6.
In vitro phosphorylation of MtlR
by proteins of the PTS. A, different combinations of
components of the B. stearothermophilus PTS; EI, HPr,
IIAmtl, and IICBmtl and MtlR were added as
indicated at the bottom. Mannitol was added during the full
experiment (lane 9) or in the last 1 min (lane
10). B, phosphorylation of MtlR by different amounts
HPr (lanes 1-4) or a 6.7-fold increased in incubation time
(lane 5). The concentrations MtlR, IIAmtl, and
mannitol, when added, were 0.8 µM, 0.08 µM,
and 10 mM, respectively. IICBmtl was added as
LGS322 vesicles to a total protein concentration of 0.12 mg/ml. In
A, the EI and HPr concentration were 1.5 µg/ml and 0.36 µM, respectively. In B, the EI concentration
was 4 µg/ml, and the HPr concentration was as indicated under each
lane. All reactions were started with 4.8 µM
[32P]PEP. The mixtures were incubated at 30 °C for 15 min, with exception of lane 5 in panel B, in
which the sample was incubated for 100 min. The reactions were stopped
with denaturation buffer and loaded on a 15% SDS-PAGE gel. The
difference in intensity between A and B is due to
the fact that these data are taken from different experiments employing
different exposure times.
1 µg
1
µM
1, respectively. The difference of a
factor of 1470 between these dependences indicates that MtlR is much
more rapidly phosphorylated by IICBmtl than by the same
concentration of HPr.
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Fig. 7.
Phosphorylation and dephosphorylation of
B. stearothermophilus MtlR B. stearothermophilus IICBmtl and HPr. The
HPr-dependent (A) and
IICBmtl-dependent (B)
phosphorylation rates of MtlR were obtained from the linear relation
between the incubation time and the intensity of the
32P-MtlR band on SDS-PAGE and were expressed as the amount
32P-MtlR formed per min per µg added MtlR. C,
time-dependent dephosphorylation of 32P-MtlR by
IICBmtl with (squares) and without
(circles) mannitol. Phosphorylation of MtlR by various
amounts of HPr was performed in 25 mM Tris-HCl, pH 7.5, 0.5 mM MgCl2, 5 mM DTT, 1 mM NaF, 4 µg/ml EI, and 6.3 µM
[32P]PEP at 30 °C. Reactions were started by the
addition of 0.8 µM MtlR. Samples were taken at various
times and analyzed by SDS-PAGE. Phosphorylation by IICBmtl
was performed under conditions similar to those used in the
phosphorylation by HPr except that IICBmtl, 0.4 µM HPr, 0.4 µM IIAmtl, and
0.25% decyl-polyethylene glycol were included in the reaction mixture.
21 µg of MtlR was first phosphorylated, as described above, for 40 min at 30 °C in 200 µl via IICBmtl in the presence of
12 µM [32P]PEP. Phosphorylated MtlR was
bound to 50 µl of Ni-NTA agarose and washed with 3 volumes of 0.5 ml
of 25 mM Tris-HCl, pH 7.5, 0.5 mM
MgCl2, 5 mM DTT, 0.25% decyl-polyethylene
glycol, and 100 mM NaCl. 32P-MtlR was eluted in
150 µl of wash buffer supplemented with 150 mM imidazole.
4 µg of 32P-MtlR was dephosphorylated by the addition of
9 nM IICBmtl in the presence
(squares) and absence (circles) of 5 mM mannitol. Samples were drawn from the reaction mixture,
mixed with loading buffer, and frozen until analysis by SDS-PAGE.
90 and
35 would contain the MtlR binding site. A DNA fragment,
containing the sequence from position
127 to +212, was labeled at the
127 end as described under "Materials and Methods." This probe
was incubated with different amounts of the purified MtlR and exposed
to DNase I digestion. Five protected areas were observed, extending
from position
41 to
86, in the presence of MtlR (Fig.
8). These areas are indeed located in the conserved regions in B. subtilis, S. carnosus,
and B. stearothermophilus, just upstream of the mannitol
promoter (Fig. 3). Hypersensitivity to DNaseI digestion, induced by the
presence of MtlR, was found at positions
70 and
48. The most
obvious hypersensitivity was found at position
70 in the presence of
0.74 µM MtlR. The decrease of intensity of this
hypersensitive band at the highest MtlR concentrations could be due to
the tendency of the protein to aggregate, which might result in
covering even the hypersensitive regions. The binding equilibria were
calculated for each of the five protected regions (data not shown). The
binding equilibrium was the same for all protected areas, indicating
that all of the areas are involved in the binding of the same regulator
complex. The MtlR concentration at which 50% protection was observed
varied from 300 to 100 nM depending on the protein sample
used.
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Fig. 8.
DNA footprint analysis of MtlR binding to the
mannitol promoter region. A DNA fragment containing the mannitol
promoter region from position 127 to +212 was synthesized by PCR,
using a 32P-labeled primer at the
127 position. In each
reaction, 20 kcpm of the DNA probe was exposed to DNase I in the
presence of 3, 1, 0.3, 0.1, 0.03, or 0.01 µg of MtlR and analyzed on
a sequence gel (lanes 1-6, respectively) (see under
"Materials and Methods" for details). Position of the binding was
determined by the A and G sequence ladder, obtained using the same
labeled primer as was used for the probe. The protected regions, FP1 to
FP5, are indicated by brackets, and hypersensitive sites HS1
and HS2 are indicated by arrows. The numbers at the
left indicate nucleotide positions relative to the mannitol
start site.
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Fig. 9.
The effect of phosphorylation on the binding
of MtlR to the mtl promoter region. For each
phosphorylation condition, MtlR, dephosphorylated by incubation at pH
6.5 and 30 °C for 2 h, was diluted to 1.3 µM in
phosphorylation buffer containing different combinations of PTS
components. The final concentrations of these components in the mixture
are listed at the top for each experiment. After 2 h of
phosphorylation at 30 °C, each mixture was diluted with the same
phosphorylation mixture without MtlR. An equal amount of DNA binding
buffer was added to each sample, resulting in final MtlR concentrations
as follows: lane 1 (of each set), 766 nM;
lane 2, 255 nM; lane 3, 77 nM; lane 4, 26 nM; lane
5, 7.7 nM; lane 6, 2.6 nM;
lane 7, 0.0 nM for each experiment.
A, DNA footprint created of each sample, as described under
"Materials and Methods." B, the increase in protection
compared with lane 7 of sets I-V,
containing no MtlR, was calculated from the intensities of the
protected region FP1 and plotted against the logarithm of the MtlR
concentration.
In vivo expression of the mannitol transporter and the
mannitol-1-phosphate dehydrogenase in B. stearothermophilus
DISCUSSION
A-dependent promoter overlapping a CRE box.
The transcription start site of this promoter was confirmed by primer
extension. Binding of the CcpA repressor to the CRE box could be
responsible for the observed catabolite repression of
IICBmtl and MPDH by glucose, as observed for similar CRE
boxes in B. subtilis (38). The similarity between the
promoter region of the mannitol operon of B. subtilis,
S. carnosus, and B. stearothermophilus and the
presence of a MtlR-like protein in all three organisms suggests a
similar mode of regulation. Binding of MtlR to a sequence upstream from
the -35 box was expected based on the similarity of this region
between these organisms. Indeed, DNA footprint experiments revealed
that MtlR binds to this region. Five regions were protected against
DNase I digestion by MtlR. It is likely that MtlR bound at this
position can affect the expression of the mannitol operon by
interacting with the RNA-polymerase binding to the promoter. The MtlR
binding site, determined by DNA footprinting, spans a large region of
46 base pairs. This could be an indication that two or more MtlR
molecules bind to this site. Because the affinity for each of the
protected areas is the same, the binding of a single complex would be
more likely than the independent or cooperative binding of several
monomers to this site.
-glucoside transporter BglF causes dissociation of the active
BglG dimer in to inactive monomers (31).
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Fig. 10.
Proposed model for the regulation of MtlR by
the PTS. MtlR can probably be phosphorylated on at least two
different domains. Phosphorylation of one domain (striped)
by HPr results in an increased affinity of MtlR for the mannitol
promoter region, whereas phosphorylation of another domain
(gray) by IICBmtl prevents binding to this
region. A, in the absence of mannitol and other substrates,
all PTS proteins will be in the phosphorylated state. MtlR will be
phosphorylated by HPr and IICBmtl and have a low affinity
for the mannitol operator, and consequently, the expression of the
mannitol operon is not stimulated. B, in the presence of
mannitol, IICBmtl is dephosphorylated by transfer of the
phosphoryl group to mannitol. The MtlR domain involved in the negative
regulation is dephosphorylated and maximal P-HPr stimulation of MtlR
binding to the operator can take place. C, when rapidly
metabolizable PTS substrates, including mannitol, are transported, the
concentration P-HPr decreases. MtlR is dephosphorylated at the
stimulation domain, and the expression of the mannitol operon is no
longer stimulated.
The mechanism coupling phosphorylation and DNA binding is still uncertain. Does phosphorylation of MtlR lead to conformational changes within the protein, changing its DNA binding domain, or does it affect the aggregation state? The observed dissociation of BglG, upon phosphorylation of PRD-I by BglF, into inactive monomers (31), favors the latter explanation. In this case, the MtlR would be a multimer that binds to the DNA as one complex, because the footprints FP1 to FP5 all have the same MtlR dependence. If phosphorylation by HPr and IICBmtl influence the oligomeric state, they will affect the concentration of the active complex in the system and possibly the affinity of this complex as well.
A 1400-fold difference between the IICBmtl and HPr dependences in the rate of phosphorylation of MtlR has been observed. Differences in phosphorylation kinetics can be expected, because the concentrations of HPr and IICBmtl in the cell are very different. These differences in phosphorylation kinetics are probably also a way to tune the interaction between the phosphorylation state of the PTS components and the activity of the transcriptional regulator. The concentration of P-his-HPr and P-IICBmtl and, consequently the phosphorylation level of MtlR will be dependent on the activity of these proteins in the PTS.
The differential effect of phosphorylation, by two different proteins,
on the DNA binding properties of a bacterial transcriptional regulator
has not, to our knowledge, been described before. Regulation of MtlR by
two components of the PTS is an example of an elegant control system
sensing both the presence of mannitol and the need to utilize this
substrate. More detailed studies on the number and locations of the
phosphorylation sites will be necessary to elucidate the suggested
negative effect of phosphorylation by HPr on a second site.
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ACKNOWLEDGEMENTS |
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We thank X. Lai and L. O. Ingram for providing us the plasmid pLOI1801 expressing B. stearothermophilus HPr and EI.
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FOOTNOTES |
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* This research was financially supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research.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.
To whom correspondence should be addressed. Fax: 31-0-503634429;
E-mail: G.T.Robillard{at}chem.rug.nl
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ABBREVIATIONS |
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The abbreviations used are:
PTS, phosphoenolpyruvate-dependent phosphotransferase system;
PEP, phosphoenolpyruvate;
EI, enzyme I of the PTS;
mtl, mannitol;
IICBmtl, mannitol permease, HPr, histidine phosphocarrier
protein;
IIAmtl, enzyme IIA of the mannitol PTS;
MPDH, mannitol-1-phosphate dehydrogenase;
IPTG, isopropyl--D-thiogalactopyranoside;
PCR, polymerase
chain reaction;
GST, glutathione S-transferase;
HTH, helix-turn-helix;
PRD, PTS regulation domain;
CRE, catabolite response
element;
PAGE, polyacrylamide gel electrophoresis;
DTT, dithiothreitol.
2 S. A. Henstra, R. H. Duurkens, G. T. Robillard, manuscript in preparation.
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REFERENCES |
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