Dimerization and DNA Binding Properties of the
Bacillus licheniformis 749/I BlaI Repressor*
Patrice
Filée
§,
Christelle
Vreuls¶,
Raphaël
Herman
,
Iris
Thamm
,
Tony
Aerts
,
Peter P.
De
Deyn
,
Jean-Marie
Frère
, and
Bernard
Joris
**
From the
Centre d'ingénierie des
Protéines, Institut de Chimie B6a, Université de
Liège, Sart-Tilman, B4000 Liège, Belgium, the
¶ Laboratoire de Physique Biomédicale, Institut de Physique
B5, Université de Liège, Sart-Tilman, B4000 Liège,
Belgium, and the
Department of Biomedical Sciences, University
of Antwerp, B-2610 Antwerp, Belgium
Received for publication, October 24, 2002, and in revised form, February 24, 2003
 |
ABSTRACT |
In the absence of penicillin, the
-lactamase
encoding gene blaP of Bacillus licheniformis
749/I is negatively regulated by the transcriptional repressor BlaI.
Three palindromic operator regions are recognized by BlaI: two in the
blaP promoter (OP1 and OP2) and one (OP3) in the promoter
of the blaI-blaR1 operon. In this study, the dissociation
constant of the purified BlaI dimer was estimated at 25 µM by equilibrium ultracentrifugation. Quantitative
Western blot analysis indicates that the intracellular concentrations
of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the
-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 µM) or in the same range as (75 µM) the
dissociation constant, respectively. This suggests that BlaI is
partially dimeric in the cytoplasm of these strains and interacts
in vivo with its operators as a preformed dimer. This
hypothesis is supported by band shift assays on an operator containing
a randomized half-operator sequence. The global dissociation constants
of the operator-BlaI dimer complexes were measured by band shift
assays and estimated as KdOP1 = 1.7 ± 0.5 10
15 M2,
KdOP2 = 3.3 ± 0.9 10
15 M2, and
KdOP3 = 10.5 ± 2.5 10
15 M2. The role of the DNA
binding properties of BlaI on the
-lactamase regulation is discussed.
 |
INTRODUCTION |
The blaP gene encodes the class A
-lactamase
of Bacillus licheniformis 749/I. In the absence of
-lactams antibiotics, the BlaI repressor prevents the transcription
of the blaP gene (1-3). Two additional genes,
blaR1 and blaR2, are also involved in the induction of the
-lactamase synthesis (4, 5). The blaP, blaI, and blaR1 genes are clustered in a
divergeon (bla divergeon) in which blaI and
blaR1 form an operon (Fig. 1).
The blaR1 gene encodes a transmembrane protein that acts as
penicillin receptor (6-8), and blaR2 is not
identified yet and is not linked to the bla divergeon. The
BlaR2 protein is essential for the inactivation of BlaI. In
Staphylococcus aureus, the blaZ and
mecA genes, encoding respectively a
-lactamase and the
low affinity penicillin-binding protein 2', are regulated by
similar elements (9, 10).

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Fig. 1.
Organization of the bla
divergeon involved in the regulation of
-lactamase synthesis in B. licheniformis
749/I and nucleotide sequence alignment of the OP1, OP2, and OP3
operators recognized by the BlaI repressor. OP1 and OP2 are
located in the blaP promoter and OP3 in that of the
blaI-blaR1 operon. , operator recognized by BlaI;
, inverted repeat sequences. The bent arrows indicate
the position of the promoter and the direction of the
transcription.
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The blaI gene encodes a 128-residue protein characterized by
two functional and separate domains (11). The DNA-binding domain, a
helix-turn-helix recognition motif, is located in the N-terminal region, and the dimerization domain is in the C-terminal region. After
deletion of the C-terminal domain, the repressor becomes unable to
dimerize and to form a stable complex with its operators. DNase
footprinting experiments and filter binding reactions revealed the
presence of three regulatory regions that are recognized specifically by BlaI (11). These operators present a 23-bp-long dyad symmetry and
show the deduced 5'-AAAGTATTACATATGTAACNTTT-3' consensus sequence (Fig.
1).
In the presence of
-lactam antibiotics, the C-terminal domain of
BlaR1 is acylated (6, 7). This event triggers the activation of the
putative cytoplasmic metalloprotease motif of BlaR1 by self-proteolysis
(8, 12). In S. aureus, the
-lactamase induction
correlates with BlaI proteolysis between residues Asn101
and Phe102, and it is postulated that the activated form of
BlaR1 proteolyses BlaI (12-15). The blaR2 gene could be
involved in this process or in the activation of BlaR1, but our recent
studies in a recombinant Bacillus subtilis harboring a
plasmid carrying the bla divergeon show contrasting results
with those in S. aureus and suggest that the inactivation of
BlaI in Bacillus is mediated by the binding of a coactivator
(16). This coactivator could maintain the BlaI dimer in a
conformation unable to bind to the DNA operators and sensitive to
cytoplasmic B. licheniformis or S. aureus
proteases. To determine the mechanism by which the coactivator
interferes with the DNA binding properties of BlaI, a detailed
analysis of the interaction pathway between BlaI and its operators was essential.
In the present study, we used purified BlaI to analyze the
multimerization of the protein by equilibrium ultracentrifugation and
its binding to the three operator sequences mentioned above by band
shift assay. The implications of the DNA binding properties of BlaI on
the
-lactamase regulation are also discussed.
 |
EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
B. licheniformis
749/I is a
-lactamase-inducible strain (7). B. subtilis
168 (ATCC 23857) was used as recipient of plasmid pDML995
(chloramphenicol resistance). Escherichia coli GI724
(Invitrogen) was used as host for plasmid pCIP152 for the
overexpression of BlaI-WT.
Plasmid pDML995 is a derivative of pMK4 (17) in which the
B. licheniformis 749/I divergeon
(blaP-blaI-blaR1) is inserted. Plasmid pCIP152 is a pLEX
derivative plasmid (Novagen) allowing the BlaI-WT overexpression
(16).
Overexpression and Purification of BlaI-WT--
The
overexpression and the purification of BlaI-WT from E. coli
GI724 transformed by the pCIP152 were performed as described previously
(16).
Fluorescent Band Shift Assay--
The fluorescent band shift
assays were performed as described previously (16, 18). The fluorescent
double-stranded oligonucleotides used in this study are presented
in Table I.
Quantitative Analysis by Western Blot--
B.
licheniformis 749/I and B. subtilis/pDML995
were grown in LB medium supplemented or not with chloramphenicol. When
the absorbance of the cultures reached 0.8 at 600 nm, the cells were harvested, and the cell titer was determined by plating dilutions. This
was repeated with several independent cultures. Cells from 10 ml of
these cultures were successively pelleted, washed with 1 ml of AQ
buffer (50 mM sodium phosphate, pH 6.8, 50 mM
KCl, 1 mM EDTA, 1 mM Pefabloc), and suspended
in 500 µl of AQ buffer before sonication (three times for 20 s).
The supernatants were collected by centrifugation, added with SDS
loading buffer, and heated for 5 min in a boiling water bath. Different
amounts of these cytoplasmic extracts were subjected to SDS-PAGE (15%)
before analysis by Western blotting. Western blots were performed using polyvinylidene difluoride membranes, anti-BlaI-WT rabbit polyclonal serum (18), goat alkaline phosphatase-conjugated anti-rabbit antibodies, and revelation with 5-bromo-4-chloro-3-indoyl phosphate and
nitroblue tetrazolium (Bio-Rad). To estimate the BlaI concentration in
cytoplasmic extracts, known quantities of purified BlaI were loaded on
the same gel. To take account of the potential loss of BlaI during the
preparation of samples, the standards were prepared according to the
following procedure: nontransformed B. subtilis cells were
pelleted, washed, and suspended in AQ buffer containing purified BlaI.
After sonication, the supernatant was collected by centrifugation,
added with SDS loading buffer, and heated for 5 min in a boiling water
bath. The blots were scanned and analyzed with the software Densito (Cybertech).
Analytical Ultracentrifugation--
Analytical
ultracentrifugation experiments were carried out in a Beckman Optima
XLA instrument at 20 °C using optical absorption detection at a
wavelength of 280 nm. The protein samples were transferred in 50 mM Hepes buffer, pH 7.6, containing 200 mM NaCl and 1 mM EDTA by dialysis, and the final BlaI
concentrations were 0.447, 0.386, 0.33, 0.276, 0.218, 0.141, and 0.076 mg/ml. Sedimentation equilibrium experiments were carried out using an
AN-60 Ti Analytical rotor with standard double-sector centerpieces and
quartz windows at 11500 rpm. Equilibrium was reached after 65 h as
determined by comparing scans taken at 5-h intervals. Equilibrated
scans were used for determining equilibrium association constants for the self-association of BlaI-WT.
Sedimentation Equilibrium Data Analysis--
Equilibrium data
were analyzed by nonlinear least squares fitting of primary absorbance
data using the Mixedfit program for Windows from L. Holladay (19, 20).
To determine the best model for describing the BlaI multimerization,
the data were fitted by using the following ideal equilibrium models:
monomer-dimer, monomer-dimer-tetramer, or isodesmic models 1, 2, and 4. The best fit was determined by examining the distributions of residuals.
 |
RESULTS |
Determination of the Dissociation Constant of BlaI Dimer by
Analytical Ultracentrifugation--
To determine the mode of
multimerization and the dissociation constant of the BlaI-WT
dimer-monomer equilibrium, sedimentation equilibrium
ultracentrifugation experiments were conducted over a large
concentration range, and experimental data were collected at 280 nm.
Typical data sets are displayed in Fig.
2. For the analysis of these data, the
measured absorbance at 280 nm versus the radial position
were fitted to mathematical functions describing various
self-association systems (monomer-dimer equilibrium,
monomer-dimer-tetramer equilibrium, and isodesmic models 1, 2, and 4).
The best fit for BlaI-WT multimerization was obtained for isodesmic
model 2 shown in Fig. 3. By using this
model, the distributions of residuals (Fig. 2B) were in good
agreement with the experimental data obtained at each protein
concentration. Moreover, the calculated quantity of protein was equal
to that introduced in the ultracentrifuge cell. Analysis of the
experimental data revealed that there were monomeric, dimeric,
tetrameric, and hexameric species in solution. Following isodesmic
model 2, the monomeric, dimeric, tetrameric, and hexameric
concentrations were calculated and are listed in Table
II. Equilibrium sedimentation experiments
showed that BlaI-WT can be present in solution as monomer, dimer,
tetramer, and hexamer in the 10-30 µM range of protein
concentration with a dissociation constant K1
estimated to 25 µM, which is the same for each
equilibrium.

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Fig. 2.
Equilibrium sedimentation data.
Centrifugation was carried out at 11,500 rpm and at 20 °C for
65 h as described under "Experimental Procedures." In
A, the measured absorbance at 280 nm versus the
radial position (distance from the center of the rotor in cm) is shown
at various concentration of protein C1 = 0.447 mg/ml,
C2 = 0.86 mg/ml, C3 = 0.330 mg/ml,
C4 = 0.276 mg/ml, C5 = 0.218 mg/ml, and
C6 = 0.141 mg/ml. B gives the residuals to
the fit expressed as the differences between the experimental data and
fitted values.
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Table II
Concentration of monomer, dimer, tetramer, and hexamer calculated by
isodesmic model 2 starting from equilibrium ultracentrifugation
data (K1 = 25 µM)
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Determination of the Intracellular Concentration of BlaI--
In
prelude to this work, the cell titers of B. licheniformis
749/I and B. subtilis/pDML995 were correlated to the
OD600 nm values of bacterial culture. Cytoplasmic
extracts from several midlog phase cultures of B. licheniformis 749/I and B. subtilis/pDML995 were
analyzed by Western blotting. The concentration of BlaI in cytoplasmic
extracts was estimated with a standard curve prepared with purified
BlaI. To take account of possible BlaI proteolysis during sample
preparation, the standard curve was obtained by the addition of
purified BlaI to pDML995-free B. subtilis cells from a
midlog phase culture and by identical preparations of cytoplasmic extracts. According to the B. subtilis cell volume
(10
15 liters) determined by Abril et al. (21),
the cytoplasmic concentrations of BlaI in B. licheniformis 749/I and B. subtilis/pDML995 were estimated at about 1.9 µM (1,100 BlaI monomers/cell) and
75 µM (45,000 BlaI monomers/cell), respectively (Fig.
4). By comparison with the dissociation
constants estimated by equilibrium centrifugation, these results
indicate that BlaI is partially present as a dimer in B. licheniformis 749/I (21% dimer) and mainly in B. subtilis/pDML995 (75% dimer).
Dithiobis(succinimidylpropionate) cross-linking experiments with
cytoplasmic extracts of B. subtilis/pDML995
yielded a similar conclusion (16).

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Fig. 4.
Quantification of the cytoplasmic BlaI
concentration in B. licheniformis 749/I and B. subtilis/pDML995 by Western blotting. A,
lane 1, cytoplasmic extract from B. licheniformis
749/I; lanes 2-6, 1.5, 5, 10, 20, and 40 ng, respectively,
of purified BlaI added with cytoplasmic extracts from nontransformed
B. subtilis. B, lane 1, cytoplasmic
extract from B. subtilis/pDML995; lanes 2-6,
1.5, 5, 10, 20, and 40 ng, respectively, of purified BlaI added with
cytoplasmic extracts from nontransformed B. subtilis.
A was overexposed to allow detection of the very low
quantity of BlaI in lane 1.
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Study of the Interaction Pathway between BlaI and Its
Operators--
Firstly, we examined the binding curve of BlaI to its
three operators. A fixed concentration of labeled operator (5 nM) was incubated with increasing concentrations of
purified BlaI (3.3 nM to 0.33 µM) for 2 h before loading onto the gels. Analysis of the electropherograms
revealed the presence of two peaks. The faster and the slower peaks
correspond to the unbound operator and the BlaI dimer-operator complex,
respectively (16). No peak corresponding to BlaI monomer-operator was
observed under these conditions. As presented in Fig.
5, a sigmoidal binding curve was observed
for each operator. For OP1, the Scatchard plot (Fig. 6) indicated a 2/1 stoichiometry for the
BlaI-operator complex, which confirms that the retarded DNA observed on
the electropherograms corresponds to the BlaI dimer-operator complex. A
Hill plot of the data obtained between 10 and 90% of OP1 saturation
(Fig. 7) yielded a Hill coefficient of 2. This value corresponds to a pathway in which two BlaI monomers
simultaneously bind the operator. This situation does not allow us to
distinguish between the dimer pathway and a highly cooperative monomer
pathway (18) (Fig. 8) characterized by a
sequential binding of monomers to each half-operator. The dimer is
formed when the second monomer interacts with the free half-operator.

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Fig. 5.
Binding curves of BlaI to its operators.
A constant concentration of fluorescent double-stranded operator (5 nM) was titrated with increasing concentrations of BlaI.
A, B, and C correspond to the binding
curves of BlaI to OP1, OP2, and OP3, respectively. The data were fitted
to: Fraction of bound DNA = [BlaI]2/([BlaI]2 + K), where
K = K2'·K3' in the monomer
pathway and K1·K1' in
the dimer pathway. [BlaI] is the free BlaI expressed as the monomer
concentration.
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Fig. 6.
Scatchard plot of the data obtained with the
OP1 operator. N is the ratio between the bound BlaI
monomer concentration and the total operator concentration.
L corresponds to the free BlaI monomer concentration.
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Fig. 7.
Hill plot of the data obtained for 10-90%
saturation of OP1. corresponds to the fraction of Bound
operator.
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Fig. 9 shows band shift assays performed
by incubating BlaI with the semi-conserved OP1 operator (half-OP1;
Table I). The half-OP1 is a 30-bp-long oligonucleotide in which the
right half-binding site of OP1 is completely degenerated. In the
presence or absence of nonspecific DNA, no complex corresponding to
BlaI monomer-half-OP1 was detected. In these conditions, only the BlaI
dimer-half-OP1 complex could be visualized, but the affinity of the
BlaI dimer for the half-OP1 was considerably reduced compared with
OP1.

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Fig. 9.
Band shift assays with the semi-conserved OP1
operator (halfOP1). A, fluorogram
obtained by incubation of BlaI with the OP1 operator in the presence of
nonspecific DNA (400-fold excess). The final concentrations of BlaI and
OP1 were 3.3 and 5 nM, respectively. The reaction mixture
was incubated at 30 °C for 2 h. B, obtained by
incubation of BlaI with the semi-conserved OP1 operator
(halfOP1) in the presence of nonspecific DNA (400-fold
excess). The final concentrations of BlaI and halfOP1 were 5.3 µM and 5 nM, respectively. C,
obtained by incubation of BlaI with the semi-conserved OP1 operator
(halfOP1) in the absence of nonspecific DNA. The final
concentrations of BlaI and halfOP1 were 88 and 5 nM,
respectively.
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In this study, all of the band shift assays were done with BlaI
concentrations well below the dissociation constant of the BlaI dimer
estimated by equilibrium ultracentrifugation. This implicates that BlaI
dimer formation could be the rate-limiting step in the BlaI-DNA binding
reaction and could be responsible for the sigmoidal binding curves.
This hypothesis is reinforced by binding time courses of BlaI to OP1.
In this assay, 16 nM BlaI and 5 nM OP1 were
incubated at 30 °C. The samples were taken after different
incubation times and submitted to the electrophoresis. Fig.
10 shows that the fraction of bound OP1
stabilizes after 2 h.

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Fig. 10.
Binding time course of BlaI to the OP1
operator. The concentrations of BlaI and OP1 were 16 and 5 nM, respectively. Nonspecific DNA (400-fold excess) was
added to the reaction. The mixture was incubated at 30 °C, and the
samples were taken after 5, 10, 15, 30, 60, 120, and 240 min.
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Analysis of the DNA Binding Activity of BlaI--
Because no BlaI
monomer-DNA complex was observed in band shift assays, it can be
concluded that a high degree of cooperativity prevails in the monomer
pathway or that the dimer pathway is valid. Under these conditions, the
fraction of bound DNA is [BlaI]2/([BlaI]2 + K1·K1') according to
the dimer pathway and [BlaI]2/([BlaI]2 + K2'·K3') according to
the highly cooperative monomer pathway. Only the global dissociation
constant K (K = K1·K1') can be
estimated on the basis of the data of Fig. 5. The values are shown in
Table III together with the
K1' values calculated on the basis of a
K1 (dimer/monomer) dissociation constant of 25 µM (see above). These results reveal that the affinity of
BlaI for the operator sequences increase in the order OP3 < OP2 < OP1.
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Table III
Equilibrium parameters of the BlaI-operator interactions
K (=K1 × K1') was
derived from the data shown in Fig. 5 on the basis of the equation
given in the text for the dimer pathway. The values of
K1' were estimated at 25 µM by
equilibrium ultracentrifugation.
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 |
DISCUSSION |
Previously, we reported that the
-lactamase induction in
B. licheniformis 749/I first requires both an
acylation of BlaR1 by a
-lactam and a
-lactam stress of the cell
(6, 16). Our studies also indicated that in B. licheniformis
749/I, the hydrolysis of BlaI during
-lactamase induction was a
consequence of its inactivation. This hypothesis was supported by the
fact that in an induced B. subtilis/pDML995 strain, BlaI
lost its DNA binding capacity but retained its ability to dimerize. In
our working model, the inactivation of BlaI occurs by interaction with
a coactivator that induces an inactive conformation of the BlaI dimer.
As a result of this conformational change, the susceptibility of the
BlaI dimer to the cytoplasmic proteases would be increased in B. licheniformis 749/I. The mechanism by which this conformational change prevents the binding of BlaI to its operators is not clarified yet.
The interaction between DNA-binding proteins and operators with a dyad
symmetry can follow two distinct mechanisms (22): the monomer pathway
and the dimer pathway (Fig. 8). The former is often cooperative and is
observed for the LexA bacterial transcriptional repressor (23, 24).
Many regulators bind DNA according to the dimer pathway (25). Because a
helix-turn-helix recognition motif interacts only with five or six base
pairs, the ability of this class of DNA-binding proteins to form dimers
and higher order oligomers is fundamental to stabilize its binding to
target DNA.
In this study, equilibrium ultracentrifugation experiments showed that
at concentrations in the micromolar range, BlaI monomers, dimers, and
tetramers were present in solution and that the BlaI multimerization
follows a pathway described by isodesmic model 2 (Fig. 3). By fitting
the experimental data, a dissociation constant of 25 µM
was found for the dissociation of the BlaI dimer, tetramer, or hexamer.
To determine the in vivo concentration of BlaI, quantitative analysis of cytoplasmic extracts were performed by Western blotting. According to these results, it appeared that the intracellular concentration of BlaI in B. licheniformis 749/I (1.9 µM) is significantly lower than the dissociation constant
of the BlaI dimer so that BlaI should be only partially dimeric in the
cell. In B. subtilis transformed by the pDML995, a high copy
number plasmid, the intracellular concentration of BlaI (75 µM), is 3-fold higher that the dissociation constant so
that multimeric forms of BlaI are predominant. This result confirms
previous cross-linking experiments in which the addition of increasing
concentrations of dithiobis(succinimidylpropionate) to
cytoplasmic extracts permitted the recovery of most of the BlaI signal
as a dimer both in the absence and presence of
-lactams antibiotics
(16). In consequence, it appears that the preformed BlaI dimer is
present in both B. licheniformis 749/I and B. subtilis/pDML995 although in very different proportions.
The fluorescent band shift assays presented in this study revealed that
the binding curves of BlaI to its operators are sigmoidal, suggesting
that the binding parameters of BlaI include two equilibria. For each
operator, the fluorograms were characterized by slower and faster bands
corresponding to free operators and BlaI dimer-operator complexes,
respectively (Fig. 9). In Scatchard plots, saturation of the operator
by BlaI was reached at a molar ratio of 2 (Fig. 6). In these
conditions, no BlaI monomer-operator complex was detected. In the
monomer pathway, this would indicate a very strong cooperativity.
Indeed, the Hill coefficient was 2, a value indicating a situation
where the binding of the two monomers to the operator is simultaneous.
Consequently, in this case, the monomer pathway and the dimer pathway
cannot be distinguished on the basis of the Hill coefficient. By using
the semi-conserved operator OP1 (half-OP1), additional arguments were
found that favor the dimer pathway. In these experiments, we observed
the formation of a complex between the BlaI dimer and half-OP1 both in
the absence and the presence of nonspecific DNA. The
absence of BlaI monomer-half-OP1 complex seems to exclude the
possibility that the binding of BlaI occurs according to the monomer
pathway. Previously, we obtained similar results by mass spectrometry
that only the BlaI dimer could bind to the OP1 operator (26) and
the semi-conserved OP1 operator.1
To substantiate the dimer pathway model, a time course of the binding
reaction was performed in conditions where BlaI was predominantly
present as a monomer ([BlaI] = 0.016 µM). The
determination of the fraction of bound DNA revealed that the
equilibrium of the DNA binding reaction was only reached after 2 h. These results underline the presence of a rate-limiting step that
could be the dimerization of BlaI.
Finally, it was also shown that BlaI recognized the three specific
operators with distinct global dissociation constants ranging from
10
15 to 10
14 M2.
Because the dissociation of the BlaI dimer was 25 µM, the
dissociation constants of the BlaI dimer-operator complexes are between
10
11 and 10
10 M. These values
are typical of prokaryotic repressors with helix-turn-helix recognition
motifs (25, 27). Comparison of the global dissociation constants
indicates that BlaI presents increasing affinities for OP3 < OP2 < OP1. In regard to the nucleotide alignments of the operators presented in Fig. 1. It appears that the OP1 operator, which
has the highest affinity for BlaI, is similar to the nucleotidic consensus sequence of the three operators of the bla
divergeon. Consequently, these results suggest that in the absence of
-lactam antibiotics, transcription from the blaP
promoter, containing the OP1 and OP2 operators, is more repressed than
that from the blaI-blaR1 promoter, which includes the OP3
operator. It has not been verified yet whether cooperative binding
occurs between the three operators of the bla divergeon.
Nevertheless, this hypothesis is supported by equilibrium
ultracentrifugation and chemical cross-linking experiments (16) that
reveal that BlaI is able to form higher order multimers. In addition,
the analysis of the intergenic region of the bla divergeon
reveals that the distance between OP1 and OP3 operators is 168 bp or 17 helical turns, indicating that these operators are on the same side of
the DNA and could be involved in a looping of the DNA.
In conclusion, it is highly probable that the repression of
-lactamase synthesis in B. licheniformis 749/I requires
preformed BlaI dimers (Fig. 11). In the
presence of inducer, activation of the metalloprotease domain of the
BlaR penicillin receptor and
-lactam stress suffered by the cell
generate a coactivator in the cytoplasm. This coactivator induces a
conformational change of the BlaI dimer that yields an inactive form of
the repressor by a mechanism similar to that described for the
inactivation of the TetR repressor involved in the tetracycline
resistance in Gram-negative bacteria (28). Indeed, the binding of
tetracycline to the C-terminal domain of the TetR repressor homodimer
induces a conformational change that increases the distance between the two N-terminal binding domains of the dimer by 3 Å, abolishing the
affinity of TetR for its operator without destabilizing the dimer.

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Fig. 11.
Model representing the inactivation pathway
of BlaI upon -lactamase induction in B. licheniformis 749/I and B. subtilis/pDML995.
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 |
FOOTNOTES |
*
This work was supported by Grant P5/33 from the Belgian
Program on Interuniversity Poles of Attraction initiated by the Federal Office for Scientific, Technical and Cultural Affaires and Fond National de la Recherche Scientifique Grant 1.5201.02, and Fonds de la Recherche Fondamentale Collective Grant 2.4530.03.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.
§
Fellow of the Fonds pour la Formation à la Recherche dans
l'Industrie et l'Agriculture.
**
Research Associate of the Fond National de la Recherche
Scientifique. To whom correspondence should be addressed. Tel.:
32-4-366-2954; Fax: 32-4-366-3364; E-mail: bjoris@ulg.ac.be.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M210887200
1
C. Vreuls and V. Gabelica, unpublished data.
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