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J Biol Chem, Vol. 274, Issue 45, 32425-32432, November 5, 1999
From the Department of Cell and Molecular Biology, Umeå
University, S-901 87 Umeå, Sweden
The Pseudomonas derived
DmpR is the specific regulator of the pVI150 plasmid encoded
dmp-operon, which encodes the enzymes of the
(methyl)phenol catabolic pathway of Pseudomonas sp.
CF600 (1). This pathway confers the ability to utilize phenol,
mono-methylated phenols, and 3,4-dimethylphenol as sole carbon and
energy sources (reviewed in Refs. 2 and 3). DmpR tightly regulates
transcription from the operon promoter, Po, resulting in expression of
the specialized catabolic enzymes only in the presence of pathway
substrates or some structural analogs (4).
DmpR belongs to the prokaryotic family of enhancer binding proteins
that exert their action on transcription mediated by RNA polymerase
utilizing the alternative sigma factor, The central C-domains are the hallmark of the family and seven (C1 to
C7) highly conserved regions within this domain have been identified
(5, 6). The C-domain contains a nucleotide binding motif and mediates
ATP binding and hydrolysis essential for transcriptional activation of,
and open complex formation by, Most In the cases of DmpR and XylR, effector specificity mutants and hybrid
proteins have genetically demonstrated that the effector response is
mediated via the A-domain (4, 13, 19). Most recently, the A-domain of
DmpR has been shown to be both necessary and sufficient to bind its
ligand phenol (20). Binding of effectors to DmpR releases its intrinsic
ATPase activity required for transcriptional activation, a process that
is mimicked by removal of the A-domain of DmpR, which results in a
derivative that has full ATPase and transcriptional promoting
activities in the absence of effectors (19). These observations,
together with genetic and biochemical evidence for specific
interactions between the A- and C-domains of DmpR (19, 20, 21), have
led to a model in which effector binding to the A-domain alleviates
interdomain repression, resulting in release of the C-domain-mediated
ATPase activity and thus promotion of transcription.
In addition to the specific regulation mechanism described above, the
DmpR-mediated regulatory circuit is also subject to physiological
regulation leading to silencing of the system in rich media until the
exponential to stationary phase transition. Recently, the global
signaling molecule (p)ppGpp has been shown to be essential for
efficient DmpR-mediated transcription from the operon promoter and to
be a molecule that links this specific regulatory circuit to the
physiological status of the cell (22).
DmpR is activated by a wide range of phenolic compounds, including
substrates of the pathway it controls and some, but not all,
structurally related compounds (4, 19). However, the magnitude of the
DmpR-mediated transcriptional response from the Po promoter varies,
depending on the position and nature of substituents on the aromatic
ring. Using a DmpR mutant derivative, DmpR-E135K, altered in its
ability to respond to pathway effectors, it has been shown that these
differences can have a profound effect on the efficiency with which the
bacteria can degrade compounds that are poor effectors of DmpR (23). In
addition to novel effector specificity, other amino acid substitutions
of residue 135 result in protein derivatives that are still
effector-responsive but can mediate varying levels of transcriptional
activation even in the absence of effectors (19). Hence, residue 135 is
located in a position that can influence both effector specificity and the A-domain-mediated repression of C-domain function.
Activation of DmpR can be divided into at least two interrelated steps:
(i) binding of the effector to a specific site(s) on the A-domain, and
(ii) alleviation of repression of the C-domain-mediated activities.
Here, we use in vivo and in vitro assays of wild
type DmpR and DmpR-E135K to determine how these two processes are
coupled for different effectors and whether different aromatic
effectors act through a single binding site on DmpR.
Plasmids--
Plasmids used for in vivo assays,
expressing DmpR (pVI401) and DmpR-E135K (pVI428) from the natural
promoter of dmpR on a broad host range RSF1010 replicon,
have previously been described (23). Plasmid pVI453 expressing the
constitutively active Luciferase Assays--
Cells were grown into exponential phase
in Luria broth (LB) (25) containing appropriate antibiotics for plasmid
selection. Because the DmpR-mediated transcriptional response in LB is
not observed until the exponential to stationary phase transition (22),
cultures were diluted and grown to A600 = 2.5 prior to exposure to the indicated concentrations of effectors for
3 h. Luciferase assays of the luxAB gene product were
performed on whole cells, using a 1:2000 dilution of decanal substrate,
as described previously (26).
Western Blot Analysis--
Crude extracts of cytosolic proteins,
SDS-polyacrylamide gel electrophoresis, transfer to nitrocellulose
filters, and Western blot analysis with polyclonal rabbit anti-DmpR
sera were as described previously (19). Antibody decorated bands were
revealed using the chemiluminescence reagents of Amersham Pharmacia
Biotech as directed by the supplier. Differences in expression levels
were assessed by comparison of dilution series of the samples and
quantification using a Molecular Dynamics densitometer.
Affinity Purification of DmpR-flag Derivatives--
Protein
expression, crude extract preparation, and immunoprecipitation of
flag-tagged proteins using Flag-M2 affinity gel (Sigma) were as
described previously (19), except for a modification in the washing
procedure. Here, after incubation with the crude extracts, bead-bound
proteins were washed three times with lysis buffer (20 mM
Tris-HCl, pH 7.5, 5 mM EDTA, 2 mM
dithiothreitol) in the presence of 0.5 M NaCl and protease
inhibitors (CompleteTM, Roche Molecular Biochemicals), followed by one
wash with lysis buffer containing 1% Triton X-100 and three washes
with 20 mM Tris-HCl, pH 7.5. 2 mM
dithiothreitol. Unlike the previously reported washing condition (19),
these modified conditions resulted in bead preparations that, when
treated with crude extract from bacteria harboring a vector control
plasmid, are devoid of detectable ATPase activity. However, the
previously reported background level of ATPase activity of DmpR-flag in
the absence of effectors still persists. A control preparation of a
mutant protein harboring a single amino acid substitution in the Walker
A motif (DmpR-G268S-flag) possesses no detectable ATPase activity in
the presence or absence of 2-methylphenol (Ref. 21 and data not shown).
Hence, the high background ATPase activity (approximately 20% of fully
activated levels) is an intrinsic property of DmpR-flag and not due to
contaminating ATPase competent proteins.
The bead-bound proteins generated as described above were resuspended
as a slurry in assay buffer (35 mM Tris-Ac, pH 7.9, 5 mM MgAc, 70 mM KAc, 20 mM
NH4Ac, 1 mM dithiothreitol) and kept on ice
prior to their use in ATPase or [14C]phenol binding
assays. The concentration of bead-bound proteins was determined by
SDS-polyacrylamide gel electrophoresis comparison of a serial dilution
of each sample against standards of known concentration and
quantification using a Molecular Dynamics densitometer.
ATPase Assays--
ATPase assays were performed at 30 °C in
60 µl of 1× assay buffer as described previously (19). Reactions
contained 1 µl of bead-bound protein and the indicated concentrations
of aromatic compound(s). Assays were initiated by the addition of
radiolabeled [ [14C]Phenol Binding Assays--
Universally
labeled [14C]phenol (5.22 Gbq/mmol) was custom
synthesized by Amersham Pharmacia Biotech. Binding assays were
performed with 10 µl of bead-bound affinity-purified flag-tagged
protein (0.95 µg of protein per µl) in a total of 200 µl of assay
buffer prepared as described above. To determine the
[14C]phenol binding capacity of DmpR-flag and
DmpR-E135K-flag, proteins were exposed to increasing concentrations of
[14C]phenol in the presence or absence of 500 µM unlabeled phenol, essentially as described previously
(20). In brief, samples were allowed to reach equilibrium at 30 °C
(<5 min under these conditions) prior to separation of protein-bound
phenol from free phenol. Separation was achieved by centrifugation
through a gradient composed of 200 µl of assay buffer layered onto
300 µl of a 40% sucrose cushion, using a bench microcentrifuge at
14,000 rpm. The radioactivity in the upper layer, containing the free
phenol, and the lower layer, containing the protein-bound phenol, was independently recovered and analyzed by scintillation counting. Specific binding of phenol was calculated by subtraction of nonspecific binding observed in the presence of 500 µM competing
unlabeled phenol. For the [14C]phenol binding competition
assays, unlabeled aromatic compounds were incubated with DmpR-flag at
room temperature for 5 min prior to addition of
[14C]phenol to a final concentration of 16 µM.
Effector Dose-dependent Activation of Transcription by
DmpR--
As a first step to address the question of what mechanism(s)
underlies the specific levels of DmpR-mediated transcription from the
Po operon promoter in response to different effectors, we made use of a
previously constructed in vivo luciferase reporter system
consisting of a single copy of Po promoter fused to the luxAB genes on the chromosome of a Pseudomonas
putida host (KT2440::Po-luxAB) and a
plasmid (pVI401) expressing wild type DmpR from its native promoter
(23). The range of effectors chosen for testing included the two best
DmpR effectors (phenol and 2-methylphenol), the two poorest DmpR
effectors (4-methyphenol and 3,4-dimethylphenol), and two noneffectors
of DmpR to which the mutant DmpR-E135K had gained the ability to
respond (2,4-dimethylphenol and 4-ethylphenol). Cultures were grown and
assayed for luciferase activity in response to up to a 3 mM
concentration of the different effectors; higher concentrations in all
cases resulted in varying degrees of inhibition (data not shown).
The results in Fig. 1 (closed
symbols) show that activation of DmpR differed both in the minimum
concentration of effector required to elicit a detectable response
(approximately a 100-fold difference: 3 µM for
2-methylphenol to 300 µM for 3,4-dimethylphenol), and
also in the shape of the activation profile. The two best effectors
(phenol and 2-methylphenol) (Fig. 1, A and B)
showed a biphasic response curve. In the case of the poor effectors, 4-methylphenol (Fig. 1C) shows a monophasic response curve,
whereas for 3,4-dimethylphenol (Fig. 1D), a plateau was not
discernible before inhibitory effects were observed. The responses to
4-methylphenol and 3,4-dimethylphenol and the first phase of the
responses to phenol and 2-methylphenol appear to be like typical
saturating binding curves, which is consistent with the previous
in vitro data of a single affinity binding site for phenol
(20). The second phase of the response to high concentrations of phenol and 2-methylphenol could be either due to a second very low affinity binding site or, more likely, due to a cellular response to these high
concentrations of the effectors. To directly test the latter possibility, we made use of the A-domain-deleted derivative
Given the above results, the data in Fig. 1 are consistent with a
single class of binding sites for each compound. However, the data
clearly dissociate sensitivity, as measured by the dose response, and
the functional output of binding, as measured by the magnitude of the
response. Phenol and 2-methylphenol elicit a maximum plateau level of
Po transcription of about 6000-7000 luciferase
units/A600 (Fig. 1, A and
B). The maximum activation level achieved using the poorest
effector (3,4-dimethylphenol, Fig. 1D) was approximately
equal to those of the plateau levels for phenol and 2-methylphenol.
Notably, however, the activation profile obtained with 4-methylphenol
reached a maximal plateau level of only 3000-4000 luciferase
units/A600 (Fig. 1C), which is
approximately 50% of the levels for phenol/2-methylphenol and the
maximum response with 3,4-dimethylphenol. This observation suggests
that the level of effector activation of DmpR involves more than a
simple direct correlation with binding affinity of the effectors, a
conclusion further substantiated by in vitro assays shown below.
Dose-dependent Activation Profiles of the Effector
Specificity Mutant DmpR-E135K--
The effector specificity mutant
DmpR-E135K was independently isolated during a genetic selection for
mutants that respond to either 2,4-dimethylphenol or 4-ethylphenol (19,
23). To shed further light on the mechanism of effector activation of DmpR, the dose-dependent effector profiles of this mutant
were compared with those of wild type DmpR using the luciferase
reporter described above and a plasmid, pVI428, expressing DmpR-E135K. Western analysis revealed that DmpR-E135K is present at levels that
were slightly lower than (approximately 75%) but still comparable to
those of wild type DmpR in the cells (Fig. 1G). At high
effector concentrations, DmpR-E135K was similar to wild type DmpR in
its activation to phenol and 2-methylphenol, had an enhanced ability to
respond to 4-methylphenol and 3,4-dimethylphenol, and had gained the
novel ability to respond to 2,4-dimethylphenol and 4-ethylphenol (Fig.
1, open symbols). The dose-dependent effector
profiles of this mutant show that 2,4-dimethylphenol and 4-ethylphenol
are almost as potent activators of DmpR-E135K as 3,4-dimethylphenol is
of the wild type protein (Fig. 1, compare D to
F). In the case of phenol and 2-methylphenol, although high
concentrations of effector elicit a similar response from both the wild
type and mutant DmpR, the shift in dose responses at lower
concentrations of effector suggests that the E135K mutation may also
influence binding affinity of these compounds.
[14C]Phenol Binding by DmpR and DmpR-E135K--
To
directly test the prediction from the in vivo experiments
described above, namely that DmpR-E135K has a lower binding affinity for phenol than the wild type protein, we used flag epitope-tagged derivatives of DmpR. The presence of the eight amino acid
carboxyl-terminal flag epitope has previously been shown to have no
effect on the protein stability or effector-mediated transcriptional
responses of DmpR in vivo (19). A system employing
[14C]phenol and affinity-purified bead-bound DmpR-flag
and DmpR-E135K-flag was used to analyze phenol binding (see under
"Experimental Procedures"). The data shown in Fig.
3 demonstrate specific and saturable
binding of phenol by these proteins. Evaluation of the binding data by Scatchard analysis gives a dissociation constant of approximately 16 µM for DmpR-flag (as found previously, Ref. 20), and a
dissociation constant of 40-65 µM for DmpR-E135K-flag.
Hence, the E135K mutant does indeed have a decreased affinity for
phenol in vitro but retains the ability to achieve maximal
activation of Po in vivo.
In Vitro Effector Binding and Response Properties of DmpR--
The
in vivo experiments outlined in Fig. 1 provide a comparison
of the responses of different protein derivatives to a given compound
and give a true reflection of the sensitivity of the response to these
compounds by the bacteria as a whole. However, interpretation of the
results with respect to binding affinities and comparison of the direct
influence of different effectors on the regulator can potentially be
complicated by factors such as (i) differential permeability of the
membrane to the different compounds, (ii) preferential uptake of some
compounds by putative active uptake systems, and (iii) as illustrated
in Fig. 2, differing global cellular responses to the different
compounds. Therefore, we employed two distinct in vitro
assays that utilize a flag epitope-tagged affinity-purified derivative
of DmpR. The first in vitro assay measures the
effector-stimulated ATPase activity of DmpR-flag to the test compounds.
The second compares the ability of the test effectors to compete for
binding of [14C]phenol at its Kd
concentration of 16 µM, to evaluate whether all effectors
bind through the same site and, if this is the case, to provide
apparent binding affinities for the different compounds.
The dose-dependent ATPase stimulation profiles of DmpR-flag
to the test effectors are shown in Fig.
4, top panels. A high basal
level of ATPase activity observed in the absence of effectors was
subtracted to obtain these values (see under "Experimental Procedures"). Surprisingly, all the effectors, including the two compounds that were not able to provoke an in vivo
transcriptional response, were found both to elicit an ATPase activity
and to be able to compete [14C]phenol binding. As a
specificity control, 3,4-dimethylbenzoate was also employed in these
assays and found to be unable to elicit an ATPase activity or compete
[14C]phenol binding (data not shown). Based on the
ability of all six test effectors to cross compete
[14C]phenol binding by wild type DmpR, it can be inferred
that they must all share a common binding site. The pathway substrate
effectors (phenol, 2- and 4-methylphenol, and 3,4-dimethylphenol)
elicit saturable activity profiles, compatible with an effector binding event being coupled to ATPase activity stimulation (Fig. 4, top panels). The concentrations of each effector required to elicit 50% of the plateau ATPase activity correspond well with the
concentrations required to inhibit 50% of [14C]phenol
binding (Fig. 4, bottom panels), i.e. binding
affinity and elicitation of an ATPase activity are commensurate.
However, importantly, binding affinity of the effector did not
correlate with the magnitude of the ATPase activity response it
elicited. For example, 4-methylphenol, to which DmpR has comparatively
high affinity, elicited slightly lower maximal ATPase activity than 3,4-dimethylphenol, to which DmpR has at least a 10-fold lower apparent
binding affinity (see Fig. 4, C, D, I, and J). As
a control for these experiments, 3 mM concentrations of all
the test compounds were found to have no effect on the ATPase activity
of
The magnitude of the effector-stimulated ATPase activities of DmpR-flag
produced with phenol, 2- and 4-methylphenol, and 3,4-dimethylphenol correspond in each case to the magnitude of the transcriptional response observed in the in vivo transcriptional activation
assay shown in Fig. 1; i.e. phenol and 2-methylphenol elicit
similar high maximal levels, 4-methylphenol elicits a lower
approximately 50% maximal level, and 3,4-dimethylphenol requires
markedly higher concentrations to elicit a response but eventually
elicits a higher level than 4-methylphenol. Thus, the level of ATPase
activity elicited by effector binding in these cases correlates with
the level of the transcriptional response, suggesting that effector binding to release the ATPase activity and functional coupling of the
ATPase activity to the transcriptional apparatus are fully co-ordinated. However, the nonpathway substrate compounds
2,4-dimethylphenol and 4-ethyphenol can elicit a low but significant
ATPase activity (Fig. 4, E and F), but not a
transcriptional response (Fig. 1, E and F).
Hence, it appears in these cases that the ATPase activity elicited by
binding of these compounds is functionally uncoupled from
transcriptional activation.
In Vitro ATPase Activity of DmpR-E135K-flag--
The ATPase
stimulation profiles in response to the test compounds were also
determined for DmpR-E135K. However, with this derivative, binding
competition assays could not be performed with any degree of confidence
due to its reduced capacity to bind [14C]phenol (see Fig.
3). Fig. 5 shows a comparison of the
effector-stimulated ATPase activities of DmpR-flag and DmpR-E135K-flag.
The dose-dependent ATPase stimulation profiles of
DmpR-E135K-flag for phenol and 2- and 4-methylphenol show profiles that
correlate with values from the in vivo transcriptional
assays; i.e. a slightly reduced ability to respond at low
concentrations of phenol and 2-methylphenol but the same maximum values
as wild type reached at high concentrations, and for 4-methylphenol, an
enhanced ability to respond in terms of the magnitude of ATPase
activity was achieved (compare Fig. 5, A-C, with Fig. 1,
A-C). In the cases of 3,4-dimethylphenol, 2,4-dimethylphenol, and 4-ethylphenol, the ATPase stimulation profiles
suggest both an increased binding affinity of DmpR-E135K (note shifts
in the curves by a 10-fold difference in effector concentration) and an
increased ability to respond as indicated by the higher ATPase activity
achieved. The increased ability to respond in terms of elicitation of
the ATPase activity by the three effectors is also observed in the
in vivo transcriptional response (see Fig. 1,
D-E). Assessment of the effect of the increased sensitivity
of the ATPase activity of DmpR-E135K to these three effectors can only
be made in the case of 3,4-dimethylphenol, because wild type DmpR lacks
an in vivo response to 2,4-dimethylphenol and 4-ethylphenol.
Comparison of the curves in Fig. 5D and Fig. 1D
show that the increased sensitivity to 3,4-dimethylphenol is not
observed as a concomitant shift in the in vivo transcription response profile. These results argue against a high threshold level of
ATPase activity being a rate-limiting step for transcriptional activation in vivo. Rather, they support the idea that some
other property of DmpR-E135K bound to these effectors, such as
conformation, reduces effective coupling of the ATPase activity to a
transcriptional response.
Competitive Inhibition of Phenol Activation of DmpR in
Vivo--
The novel response abilities of DmpR-E135K could be
envisioned to occur by two mechanisms: (i) acquisition of a novel
ability to bind the effectors, or (ii) modulation of a preexisting but nonproductive ability to bind the compounds. The above finding, namely
that the E135K mutation influences to varying degrees the response to
all six effectors tested, suggested that in this derivative, all the
effectors may mediate their action through the same binding site. The
[14C]phenol binding competition results shown in Fig. 4
demonstrate that 2,4-dimethylphenol and 4-ethylphenol can compete
phenol binding, albeit inefficiently, on wild type DmpR. These results
therefore, support the idea that the novel effector specificity of
DmpR-E135K has evolved through modulation of the functional outcome of
binding these compounds through its phenol-binding site. Such a
mechanism would predict that 2,4-dimethylphenol and 4-ethylphenol
should act as competitive inhibitors of phenol-stimulated DmpR in
functional activity assays. To test this prediction, we performed
competition assays for both in vivo transcription, using the
KT2440::Po-luxAB (pVI401) reporter system (Fig.
6A), and in vitro
ATPase activity using DmpR-flag (Fig. 6B). In each case,
phenol was present at a fixed concentration that elicited maximal
levels of activity when present alone, and challenged with increasing
concentrations of the two test compounds (Fig. 6, closed
symbols). For comparison, the activity profiles elicited by
2,4-dimethylphenol or 4-ethylphenol alone are also shown (Fig. 6,
open symbols). Both compounds were able to inhibit
phenol-mediated activation when present in the 0.1-3 mM
range, i.e. the same concentration range in which they competed [14C]phenol binding by wild type DmpR in
vitro and in which an in vivo transcriptional response
is seen by DmpR-E135K (Figs. 1 and 4, compare E and
F in both figures). As controls for these experiments, the
effects of the same concentration range of the test compounds on
activity of the constitutively active effector-independent A-domain-deleted derivatives of DmpR, The aromatic-responsive regulator DmpR and its close relatives are
unusual in that they have to directly respond to the presence of the
multiple compounds that serve as substrates for the pathways that they
control. As outlined in the Introduction, the consequences of
interaction of DmpR with the aromatic effectors in terms of the level
of transcription and expression of the specialized catabolic enzymes
can be a major limiting factor for growth at the expense of some
compounds (23). Here, we dissect the different steps by which phenolic
compounds control DmpR-driven transcription.
The A-domain of DmpR is both necessary and sufficient to bind phenol
(20). Based on cross competition of [14C]phenol binding
and phenol-stimulated functional assays (Figs. 4 and 6), all six
effectors tested here share a common binding site. Because these
effectors span the range from the best to the poorest effector of DmpR
and also include two compounds that are unable to elicit a
transcriptional response, this result strongly supports the idea that
all (>20 known) DmpR effectors mediate their action through a single
binding site within the A-domain of DmpR. Comparison of the binding
affinities with the ATPase activity elicited by the different effectors
(Fig. 4) revealed that the binding affinity of DmpR for the effectors
determined the approximate 100-fold difference in sensitivity of the
response of DmpR to the different compounds. However, the magnitude of the ATPase activity and transcriptional response elicited by the different effectors was independent of binding affinity (Fig. 1 and 4).
Hence, another factor(s) must play a role in determining the functional
outcome of binding in terms of its regulation of the levels of these activities.
Residue 135 has been shown to be intimately involved in effector
response of both DmpR (19, 23) and its close relative XylR (13, 27).
The E135K mutation of DmpR imparts the ability to mediate a
transcriptional activation in response to 2,4-dimethylphenol and
4-ethylphenol. Direct assessment of the binding ability of DmpR-E135K
for the novel effectors was not possible. However, the
dose-dependent ATPase activity profiles indicated both an increased binding affinity for the two compounds and an increased ability for these compounds to elicit an ATPase activity (Fig. 5). As
outlined in the Introduction, other substitutions of Glu-135 (E135A and
E135D), in addition to conferring novel effector specificity, also
weaken the A/C-domain repressive interaction, resulting in a
semiconstitutive phenotype in the absence of effectors. Therefore, although the E135K substitution does not result in a detectable semiconstitutive phenotype, the E135K mutation may also modulate the
A/C-domain interaction and thus enhance the maximal response to the
novel compounds. Both DmpR and DmpR-E135K can bind both 2,4-dimethylphenol and 4-ethylphenol. However, unlike wild type DmpR,
DmpR-E135K is able to couple at least part of the ATPase activity
elicited to an in vivo transcriptional response (Figs. 1 and
4). Hence, the novel effector specificity of DmpR-E135K appears to be
derived primarily by modulation of functional consequences of binding
the compounds, rather than acquisition of a novel binding capacity.
Deletion of the regulatory A-domain of DmpR and many other
We thank R. Dixon and M. Gullberg for
fruitful discussion and M. Gullberg for critical reading of the
manuscript. We also acknowledge L. C. Ng for preliminary in
vivo transcription assays and Eleonore Skärfstad for skilful
technical assistance.
*
This work was supported by grants from the Swedish Research
Councils for Natural and Engineering Sciences, the Swedish Foundation for Strategic Research, and the J. C. Kempe Foundation.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.
1
E. O'Neill, C. C. Sze, and V. Shingler,
unpublished data.
Novel Effector Control through Modulation of a Preexisting
Binding Site of the Aromatic-responsive 
54-Dependent
Regulator DmpR*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
54-dependent DmpR activator regulates
transcription of the (methyl)phenol catabolic dmp-operon.
DmpR is constitutively expressed, but its transcriptional promoting activity is positively controlled in direct response to the presence of
multiple aromatic effectors. Previous work has led to a model in which
effector binding by the amino-terminal region of the protein relieves
repression of an intrinsic ATPase activity essential for its
transcriptional promoting property. Here, we address whether the
observed differences in the potencies of the multiple effectors (i)
reside at the level of different aromatic binding sites, or (ii) are
mediated through differential binding affinities; furthermore, we
address whether binding of distinct aromatic effectors has different
functional consequences for DmpR activity. These questions were
addressed by comparing wild type and an effector specificity mutant of
DmpR with respect to effector binding characteristics and the ability
of aromatics to elicit ATPase activity and transcription. The results
demonstrate that six test aromatics all share a common binding site on
DmpR and that binding affinities determine the concentration at which
DmpR responds to the presence of the effector, but not the magnitude of
the responses. Interestingly, this analysis reveals that the novel
abilities of the effector specificity mutant are not primarily due to
acquisition of new binding abilities, but rather, they reside in being
able to productively couple ATPase activity to transcriptional
activation. The mechanistic implications of these findings in terms of
aromatic control of DmpR activity are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
54
(N) that recognizes 
24 GG, 12 GC promoters (reviewed
in Ref. 5). DmpR, like other family members, has a distinct domain
structure consisting of an amino-terminal signal reception A-domain
linked to a central activation C-domain by a short B-domain, and a
carboxyl-terminal D-domain that contains a helix-turn-helix DNA binding motif.
54-RNA polymerase (7, 8).
Mutational and cross-linking studies have also implicated the conserved
C3 region of this domain in direct interaction with the transcriptional
apparatus through the 
- and 54-subunits of the
holoenzyme (9, 10).
54-dependent activators are
constitutively produced, but their activity is controlled in response
to environmental signals. Distinct activation mechanisms are mediated
through modulation of the A-domains of the regulators and include
phosphorylation cascades, ligand binding, and signal-responsive
protein-protein interactions (reviewed in Ref. 11). DmpR belongs to a
now extensive subgroup of ligand-responsive regulators that also
includes the aromatic-responsive regulators XylR (12, 13), PhhR (14), TbuT (15), and MopR (16); the formate-responsive FhlA (17); and the
ornithine/citrulline-responsive RocR (18).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A2-DmpR derivative from the Ptac
promoter of the broad host range RSF1010-based vector, has also been
previously described (19). T7 promoter plasmids were used for high
level expression of DmpR derivatives harboring a carboxyl-terminal
fusion of the eight amino acid flag epitope tag (24). Plasmids for the
expression DmpR-flag (pVI456) and A2-DmpR-flag (pVI457) have
previously been generated (19). Analogous plasmids, pVI538 and pVI539,
for expression of DmpR-E135K-flag and DmpR-G268S-flag, respectively,
were constructed by replacing the NdeI to StuI
fragment of pVI457 (spanning the ATG initiation codon to within the
C-domain) with the NdeI to StuI fragments from
plasmids expressing these mutant proteins from Ptac
promoter vectors (21, 23).
-32P]ATP (Amersham Pharmacia Biotech)
that had been diluted (1:9) with unlabeled ATP (Amersham Pharmacia
Biotech) and added to a final concentration of 3 mM. Rates
of ATP hydrolysis at each concentration of effector was determined by
removing aliquots at 10-min intervals over 1 h, during which the
reaction was linear. Samples were adjusted to 0.1% SDS, boiled for 1 min, spotted onto polyethyleneimine cellulose plates (Merck), and
dried, and the free phosphate was separated from ATP by ascending
chromatography in 0.75 M potassium hydrogen phosphate
buffer, pH 3.5. Radioactivity was quantified using a Molecular Dynamics
PhosphorImager. The value for phosphate released was calculated as a
percentage of the total amount of radioactivity per sample.
Effector-stimulated ATPase hydrolysis was calculated by subtraction of
a basal ATPase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A2-DmpR. This derivative is constitutively active both in vivo and
in vitro and thus bypasses the need for the aromatic
activation step (19). Plasmid pVI453 carries A2-dmpR
downstream from the
isopropyl--D-thiogalactopyranoside-inducible promoter of
the vector. This plasmid was introduced into the reporter strain
P. putida KT2440::Po-luxAB and cultured
in the presence of 0.5 mM
isopropyl--D-thiogalactopyranoside to produce A2-DmpR at approximately the same level as wild type DmpR produced from its
native promoter (19). The level of transcription achieved in the
presence of aromatic compounds was monitored as luciferase activity. As
shown in Fig. 2A, low levels
of 2-methylphenol did not affect the level of transcription mediated by
this constitutively active derivative, whereas exposure to higher
concentrations (1-3 mM) resulted in higher levels of
transcription. The level of transcription mediated by A2-DmpR in the
presence of 3 mM concentrations of the six test effectors
(and a control compound, 3,4-dimethylbenzoate, that does not elicit a
response from wild type DmpR) is shown in Fig. 2B. The
various aromatic compounds had different effects on the transcriptional
activity ranging from essentially no effect (4-methylphenol) to a 40%
enhancement of luciferase activity (3,4-dimthylphenol). Most
importantly, 3 mM concentrations of the two effectors that showed a second phase in the dose-dependent response curves
(phenol and 2-methylphenol, Fig. 1, A and B),
showed marked enhancement of the A2-DmpR-mediated transcription,
whereas a 3 mM concentration of 4-methylphenol that
resulted in a monophasic dose-response curve (Fig. 1C) had
little or no effect on A2-DmpR activity (Fig. 2B). Hence,
the effect of the different compounds on global cellular response
primarily accounts for the second phase of the response seen with
phenol and 2-methylphenol but not 4-methylphenol. Recently, (p)ppGpp
has been shown to be required for, and to enhance, maximal levels of
DmpR-mediated transcription from Po (22). Therefore, elevation of
cellular concentrations of (p)ppGpp in response to high concentrations
of phenol and 2-methylphenol is a likely mechanism underlying the data
shown in Fig. 2.
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Fig. 1.
In vivo mediated transcriptional
response of Po by DmpR (closed symbols) and DmpR-E135K
(open symbols). A-F, the luciferase
transcriptional response of P. putida
KT2440::Po-luxAB harboring plasmids expressing
DmpR (pVI401) or DmpR-E135K (pVI428) was measured in the absence or
presence of increasing concentrations of effector as described under
"Experimental Procedures." Shown are the averages of triplicate
determinations in each of two to four independent experiments;
bars indicate standard error. G, expression
levels of DmpR and DmpR-E135K. Western analysis of 30 (lanes
1 and 3) and 15 (lanes 2 and 4)
µg of crude extract derived from cells used in A-F were
separated by 11% SDS-polyacrylamide gel electrophoresis and treated
with anti-DmpR as described under "Experimental Procedures."
View larger version (28K):
[in a new window]
Fig. 2.
In vivo mediated transcriptional
response of Po by A2-DmpR. A,
the luciferase transcriptional response of P. putida
KT2440::Po-luxAB harboring pVI453 expressing
A2-DmpR in the presence of increasing concentrations of
2-methylphenol. The dashed line shows the profile of wild
type DmpR for comparison. B, luciferase activity of P. putida KT2440::Po-luxAB (pVI453) in the
presence of 3 mM of the indicated aromatic compound.
Conditions were as under Fig. 1, except that 0.5 mM
isopropyl--D-thiogalactopyranoside was included to
induce transcription from the plasmid-located Ptac
promoter. Shown are the averages of triplicate determinations in each
of two independent experiments; bars indicate standard
error. dmb, dimethylbenzoate; dmp,
dimethylphenol; ep, ethylphenol; mp,
methylphenol.
View larger version (22K):
[in a new window]
Fig. 3.
Comparison of [14C]phenol
binding of DmpR-flag (closed symbols) and
DmpR-E135K-flag (open symbols). Data are the
average of triplicate determinations of two independent experiments.
Top inset shows a Coomassie Blue stain of proteins released
from 3 µl of the beads and separated by 11% SDS-polyacrylamide gel
electrophoresis. Bottom inset shows Scatchard analysis used
to derive the dissociation constants for DmpR-flag
(Kd = 16 µM) and DmpR-E135K-flag
(Kd = 40-65 µM) for phenol.
A2-DmpR-flag (data not shown), ruling out complications of
potential toxic effects of specific compounds on ATPase activity
per se. Hence, the binding affinity directly correlates to
the sensitivity with which DmpR responds with ATPase activity to the
presence of the effector, but it can be dissociated from the magnitude
of this response.
View larger version (19K):
[in a new window]
Fig. 4.
In vitro properties of
DmpR-flag. Dose-dependent stimulation of the ATPase
activity of DmpR-flag (A-F) and competition of 16 µM [14C]phenol binding by the different
effectors (G-L) were determined as described under
"Experimental Procedures." The data in A-F are the
average of two to four independent experiments and are expressed as a
percentage of the maximal ATP hydrolysis rate elicited by the best
effector 2-methylphenol (103 nmol of ATP hydrolyzed min 
1
nmol1 DmpR-flag dimer). Values in G-L are the
averages of triplicate determinations and are expressed as a percentage
of [14C]phenol bound in the absence of any competitor.
Dashed lines indicate 50% stimulation or competition where
plateau levels are discernible.
View larger version (25K):
[in a new window]
Fig. 5.
In vitro
dose-dependent ATPase activity stimulation of
DmpR-E135K-flag by different effectors. The data are the average
of two to four independent determinations and are expressed as a
percentage of the maximal ATP hydrolysis rate elicited by the best
effector 2-methylphenol (103 nmol of ATP hydrolyzed min 1
nmol1 DmpR-flag dimer). The profiles for wild type
DmpR-flag are shown as dotted lines to aid comparison.
A2-DmpR, and A2-DmpR-flag were monitored. Because no effect of 2,4-dimethylphenol or
4-ethylphenol was seen on the activity of these derivatives (data not
shown), we conclude that the decreased transcription and decreased
ATPase activities observed with increasing concentrations of these two compounds represent true competitive inhibition through the same binding site as phenol. Given that these two compounds compete both
[14C]phenol binding and phenol-stimulated activity on
wild type DmpR, the results further argue that the E135K mutation
recouples the ATPase response to 2,4-dimethylphenol and 4-ethylphenol
binding to transcriptional activation that is functionally uncoupled in wild type DmpR.
View larger version (21K):
[in a new window]
Fig. 6.
In vitro and in vivo
competitive inhibition of phenol-stimulated activities of DmpR by
2,4-dimethylphenol (circles) and 4-ethylphenol
(triangles). A, the luciferase
transcriptional response of P. putida
KT2440::Po-luxAB (pVI401) was measured in the
presence on 30 µM phenol and the indicated concentrations
of competitor (filled symbols). The profiles of
transcriptional activation elicited by 2,4-dimethylphenol or
4-ethylphenol alone are also shown for comparison (open
symbols). Shown are the averages of triplicate determinations from
two independent experiments, expressed as a percentage of the response
elicited by 30 µM phenol alone. B, DmpR-flag
was assayed for ATPase activity in the presence of 0.3 mM
phenol and the indicated concentrations of competitor (filled
symbols). The profiles of ATPase activity stimulation elicited by
2,4-dimethylphenol or 4-ethylphenol alone are also shown for comparison
(open symbols). Shown are the averages of duplicate
determinations, expressed as a percentage of the maximal response
elicited by 2-methylphenol (103 nmol of ATP hydrolyzed
min 1 nmol1 DmpR-flag dimer).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
54-dependent regulators results in
signal-independent derivatives possessing high intrinsic ATPase
activity and the ability to promote transcription (19, 28-33). These
observations suggest that, with the exception of NtrC, the regulatory
A-domains do not serve any activating function per se
(reviewed in Ref. 11). Indeed, the A-domain-deleted derivative of DmpR
possesses ATPase and transcriptional promoting activities comparable to
those of DmpR fully activated by its best effector (Fig. 2) (19). Thus,
binding of the different effectors by the A-domain controls the
activity of DmpR by alleviating repression of both its
C-domain-mediated activities, namely its intrinsic ATPase activity and
productive interaction with the transcriptional apparatus. Previous
analysis using only the best effectors of DmpR had suggested a model
whereby binding of phenol by the A-domain of DmpR controls hydrolysis
of ATP, which, because it is a prerequisite for transcriptional
activation, in turn controls the level of the transcriptional promoting
property of DmpR (19, 20). However, whether this control is exerted
directly, by blocking access of ATP, or indirectly by some other
mechanism, such as control of the oligomeric state of the protein,
remains to be elucidated (11). The finding that purified DmpR has high
basal ATPase activity even in the absence of effector suggests that ATP
has a degree of access to its binding site on DmpR under all conditions. The results shown here with the novel effectors of DmpR-E135K, and the poorer effectors of DmpR, suggest that binding of
different aromatic effectors may result in either complete or only
partial alleviation of the inhibitory action of the A-domain. Furthermore, they also suggest that partial alleviation has the potential to result in differential effects on ATPase activity and its
productive coupling to the transcriptional apparatus, i.e.
that regulation of these two processes can under some circumstances be
dissociated. With pathway substrate effectors, the different levels of
ATPase activity elicited from DmpR in vitro correlated well
with the levels of the DmpR-mediated transcriptional response in
vivo (Figs. 1 and 4). This is exemplified by 4-methylphenol to
which DmpR has relatively high affinity but that is only capable of
eliciting approximately half maximal ATPase and transcriptional response (Figs. 1 and 4). This correlation suggests that in some cases,
the levels of ATPase activity directly determine the level of
transcriptional initiation, and control of ATPase activity and
productive interaction with the transcriptional apparatus by effector
binding is co-ordinated. However, in the case of 2,4-dimethylphenol and
4-ethylphenol, elicitation of an ATPase activity is uncoupled from
transcriptional activation. The in vitro ATPase stimulation assays are performed in the absence of the transcriptional apparatus, and it is not known how much of the ATPase activity elicited would be
productively coupled to transcriptional activation in vivo. The mechanism by which ATPase activity of the C-domain is coupled to
transcriptional activation is complex and as yet not fully understood.
ATP binding per se has been shown to mediate conformational changes that promote oligomerization required prior to ATP hydrolysis (34-37). Mutations within the C3 region, required for productive interactions with 54-holoenzyme, that uncouple ATPase
activity from transcriptional activation have been found for NtrC (38),
DctD (10), and DmpR.1
Similarly, a mutation in a different location of the central domain of
NtrC, within the proposed ATP binding pocket, has also been shown to
uncouple ATP hydrolysis from transcriptional activation (39). Thus,
exposure and correct presentation of both the ATP binding site and the
transcriptional apparatus interactive surface must be important for
productive coupling of the catalytic activity via protein-protein
interactions. Thus, for DmpR, it would appear plausible that A-domain
binding of certain aromatic compounds may mediate conformational
changes that allow access (and thus hydrolysis) of a small ATP molecule
but still mask regions of the C-domain necessary for productive
protein-protein interaction with the transcriptional apparatus. Hence,
we would speculate that for productive effectors, binding of the
aromatic compound to the A-domain unmasks C-domain regions involved in
both these processes and that the defect in coupling 2,4-dimethylphenol
and 4-ethylphenol stimulated ATPase activity with transcriptional activation resides in the conformation/oligomeric state adopted by DmpR
upon binding of these compounds. Testing the possible mechanisms
outlined above using purified components and in vitro transcription assays is a future focus of our laboratory.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 4-6-90-7852534;
Fax: 46-90-771420; E-mail: victoria.shingler@cmb.umu.se.
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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