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Journal of Bacteriology, February 2004, p . 1200-1204, Vol .
186, No . 4
Benzoate Decreases the Binding of cis,cis-Muconate to the BenM
Regulator despite the Synergistic Effect of Both Compounds on Transcriptional
Activation
Todd J . Clark,1,
Robert S . Phillips,2 Becky M . Bundy,1,
Cory Momany,3 and Ellen L . Neidle1*
Departments of Microbiology,1 Chemistry, and Pharmaceutical,2
Biomedical Sciences, University of Georgia, Athens, Georgia 306023
Received 9 April 2003/ Accepted 3 November 2003
Fluorescence emission spectroscopy was used to investigate interactions
between two effectors and BenM, a transcriptional regulator of
benzoate catabolism . BenM had a higher affinity for cis,cis-muconate
than for benzoate as the sole effector . However, the presence
of benzoate increased the apparent dissociation constant [reduced the
affinity] of the protein for cis,cis-muconate . Similar results
were obtained with truncated BenM lacking the DNA-binding domain .
High-level transcriptional activation may require that some
monomers within a BenM tetramer bind benzoate and others bind cis,cis-muconate .
BenM, a member of the large and diverse family of LysR-type
transcriptional regulators, controls benzoate degradation by the soil
bacterium Acinetobacter sp . strain ADP1 [7,
20] . BenM belongs to a LysR-type subfamily
controlling the bacterial catabolism of aromatic compounds, including
pollutants [6, 8, 12,
17-19, 22,
23] . Many regulators in this subfamily bind cis,cis-muconate
[hereafter designated muconate] or halogen-substituted muconates
as effectors . BenM additionally responds to benzoate, a compound
that generates muconate during its catabolism [depicted in Fig .
1] . Furthermore, benzoate and muconate together have a
synergistic effect on transcriptional activation [1] .
Thus, it seems likely that both compounds bind simultaneously to a
BenM tetramer . Effector-dependent transcription and DNA-binding
properties have been characterized for several muconate-responsive
proteins [1, 3, 14,
15, 18] . However, there have been no
direct studies of their interactions with effectors . Here,
fluorescence emission spectroscopy was used to investigate the
interactions of BenM with benzoate and muconate . These studies raise
the possibility that effector-dependent conformational changes in the
tetrameric form of BenM provide the basis for transcriptional
synergism .
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FIG . 1 . BenM responds to benzoate and its catabolite cis,cis-muconate .
BenM activates the expression of genes coding for BenABC, BenD, and
CatA, the enzymes that convert benzoate to muconate . The degradation of
muconate to tricarboxylic acid cycle intermediates enables benzoate to
serve as a sole carbon source for strain ADP1 [7].
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To confirm the function of the region postulated to interact with
effectors, a truncated BenM derivative was generated that lacks 80
N-terminal amino acids . The removal of this N-terminal region may
facilitate structural studies in a fashion similar to that observed
for OxyR and CysB, two distantly related LysR-type regulators [2,
21] . As reported here, we determined the affinities
of muconate and benzoate for BenM and the engineered protein,
which was designated BenM-EBD after proving to encompass the
effector-binding domain of the regulator .
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BenM-EBD lacks the putative DNA-binding domain .
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Residues 18 to 37 of BenM are predicted to form a DNA-binding
helix-turn-helix structure [10, 16,
20] . To remove this region, a benM segment
was amplified with two PCR primers, 5'-TCAATTCATATGACCAAGCGCATTGCC-3'
[BenM-81] and 5'-TCAATTCTCGAGCCAGTTTGGCGGCTCAGTAAA-3' [BenM-3-Xho],
and inserted into vector pET21b [Novagen] by using the engineered
[underlined] restriction sites NdeI and XhoI . The benM
segment was subsequently expressed from plasmid pBAC435 to produce a
protein in Escherichia coli missing 80 amino acids at the N
terminus of BenM . This BenM-EBD protein contained a six-histidine
tag at the C terminus to facilitate protein purification . Two
amino acids, leucine and glutamate, were also added between the
native C terminus and the histidine tag . The methods used to generate
and purify this protein were similar to those used for the
histidine-tagged full-length BenM protein [1, 4] .
BenM-EBD was obtained at greater than 95% purity, as assessed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Gel
filtration and liquid chromatography-mass spectrometry methods
indicated that BenM-EBD is a dimer in solution [5]
while the full-length protein is a tetramer in solution [1] .
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Similarity between BenM and BenM-EBD in interactions with
benzoate . |
BenM-EBD was compared to the full-length regulator by fluorescence
emission spectroscopy, a sensitive technique for detecting interactions
that directly or indirectly alter the environment of tryptophan
residues [11] . BenM and BenM-EBD each have a single
tryptophan that precedes a histidine purification tag . This
tryptophan constitutes the C terminus of the 304-amino-acid native
regulator . The histidine tag of the full-length protein does not
alter the regulatory function in vitro or in vivo [1],
and the designation BenM here refers to the histidine-tagged
full-length protein used in these studies .
The effects of benzoate on the fluorescence emission spectra of
the proteins were determined with an excitation wavelength of 280 nm
[Fig . 2A and B] . The spectrofluorimeter used [RSM-1000;
OLIS, Inc.] has a 450-W Xe lamp with an 8-nm band pass for the
exit slit, on the excitation monochromator . The band passes for the
entrance and scan disk slits, respectively, of the emission
monochromator are 4 and 6 nm . Samples of BenM or BenM-EBD were
diluted to a final monomeric concentration of 2 µM with buffer [20 mM
Tris-HCl [pH 7.9], 500 mM NaCl, 10% glycerol] . Effectors [2 to 10 µl]
were added to the protein solutions to achieve final concentrations
in the range of 10 µM to 10 mM in a total reaction volume of 2 ml .
The fluorescence emission was scanned at 25°C for 30 s with 31
scans/s over a wavelength range of 300 to 900 nm . Data were analyzed
by curve fitting to a single species by using global analysis
software provided by OLIS [13] .
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FIG . 2 . Fluorescence emission spectra of BenM and BenM-EBD . Emission
intensities were assessed by using voltage measurements from the
spectrofluorimeter [y axes] . Vertical lines indicate the
wavelengths corresponding to the maximum intensities of the spectra .
From the top to the bottom of panels A and B, the spectra correspond to
protein samples with 0, 0.2, 0.6, 2, 4, and 8 mM benzoate with an
excitation wavelength of 280 nm . From the top to the bottom of panels C
and D, the spectra correspond to protein samples with 0, 0.04, 0.08,
0.2, 0.4, and 0.8 mM muconate with an excitation wavelength of 295 nm .
Each spectrum was compiled from the average values of at least three
separate experiments . The standard deviations of the averages were less
than 20% of the corresponding values . In the insets, the change in
spectrum intensity, measured at 340 nm [panels A and B] or at 332 nm
[panels C and D], was plotted versus the amount of effector . This change
was determined as the initial fluorescence [Fo] minus the
fluorescence at concentration i of the effector [Fi] . In
these plots, the curves were fitted to a hyperbolic binding isotherm
[Table 1].
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The top spectrum in each panel corresponds to the emission of the
protein without effectors [Fig . 2] . Addition of benzoate
to BenM or BenM-EBD caused a concentration-dependent decrease
in the intensity of the spectrum [panels A and B] . This result
suggests that the sole tryptophan residue is in or near the
effector-binding site or that benzoate causes a conformational change
in the protein . The intensity change versus the benzoate
concentration was plotted [panel insets], and the data matched well
with curves fitted to a hyperbolic regression function [for the
equation used, see Table 1, footnote a] . The shape
of the curves indicated that different protein monomers had the
same affinity for benzoate . There was no indication of cooperative
binding of benzoate to either the BenM tetramer or the BenM-EBD
dimer . The affinity of each protein for benzoate was determined [11] .
With BenM or BenM-EBD, half-maximal fluorescence quenching was
achieved with approximately 1 mM benzoate, a value that represents
the dissociation constant for this effector [Table 1] .
| TABLE 1 . Effector dissociation constantsa for BenM and
BenM-EBD
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The maximum fluorescence emission intensity occurred at approximately
332 nm for each protein in the absence of effectors . This maximum
shifted to higher wavelengths as the concentration of benzoate
increased [vertical lines in Fig . 2] . This red shift is
indicative of the tryptophan residue being exposed to a more
hydrophilic environment in the presence of benzoate [9] .
Thus, benzoate appears to alter the local conformation of the
tryptophan in both BenM and BenM-EBD and may indicate that more
substantial structural changes occur .
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Differences between the effects of muconate and benzoate
on BenM and BenM-EBD .
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The strong absorbance of muconate at 280 nm precluded the use of this
excitation wavelength; hence, an excitation wavelength of 295 nm was
used to study muconate-protein interactions [Fig . 2C
and D] . Muconate, like benzoate, quenched the fluorescence in a
concentration-dependent fashion consistent with muconate binding to
BenM and BenM-EBD . As was found for benzoate, the data fit hyperbolic
curves [insets] well, indicating the absence of cooperativity in the
binding of muconate to different protein subunits . It required less
muconate than benzoate to achieve a comparable reduction in
fluorescence intensity, indicating that BenM and BenM-EBD have higher
affinities for muconate than for benzoate [Table 1] .
Unlike benzoate, muconate failed to shift the wavelength maximum
of the emission spectrum from 332 nm, regardless of the muconate
concentration [vertical lines in Fig . 1C and D] . The absence
of a shift suggests that muconate binding does not significantly
alter the polarity of the tryptophan environment . Thus, the
benzoate-induced alteration of the protein conformation appears
distinct from muconate-induced effects on BenM and BenM-EBD .
Furthermore, while both proteins had similar affinities for benzoate,
BenM-EBD had approximately twice the affinity for muconate that the
full-length regulator did [Table 1] . While the
basis for the higher affinity of BenM-EBD for muconate is not
evident, the truncated protein clearly interacts with both effectors .
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Binding of muconate to BenM in the presence of benzoate.
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To investigate simultaneous interactions between the two effectors
and a regulatory protein, the affinity of BenM [or BenM-EBD] for
muconate was assayed in the presence of 1 mM benzoate [approximately
equal to the dissociation constant for this effector] . The ability of
benzoate to increase the affinity of BenM for muconate would be
indicative of cooperativity . Instead, benzoate decreased the apparent
affinity of BenM and BenM-EBD for muconate by a factor of 2 [Fig.
3 and Table 1] . This result is consistent
with competitive binding of the two effectors, although the
data do not rule out the possibility that the two compounds bind to
different sites on the regulatory protein . Previous studies failed to
detect any cooperative effects of the two compounds on transcription
in vitro [1] . A small amount of one compound does
not boost the activation abilities of the other . Rather, maximal
transcriptional activation occurs with approximately equimolar
amounts of both compounds [1] .
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FIG . 3 . Benzoate caused the apparent binding affinity of BenM and
BenM-EBD for muconate to decrease . Graphs were plotted from spectral
data as described for Fig . 2 . Spectral changes were
measured at 332 nm for muconate alone or at 340 nm when benzoate was
present . Dissociation constants derived from these data are shown in
Table 1.
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A previous regulatory model based on DNase I footprinting and in
vitro transcription studies [1] was modified in accordance
with the results of fluorescence emission spectroscopy . As depicted
in Fig . 4, a BenM tetramer bound solely to muconate
[lower left corner] has a different conformation than when
interacting solely with benzoate [upper right corner] . Consistent
with this model, benzoate, but not muconate, altered the wavelength
of the fluorescence emission maximum of BenM [Fig . 2] .
The different conformations of the tetramer could affect contacts
with RNA polymerase and account for the ability of muconate to
activate BenM-dependent benA transcription to a higher maximal
level than benzoate does [1] .
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FIG . 4 . Model of BenM-regulated expression of benA . The levels of
transcriptional repression or activation and the positions of BenM
dimers on three DNA-binding sites [sites 1, 2, and 3] were determined by
DNase I footprinting and in vitro transcription assays [1] .
A previous regulatory model was modified here to indicate that
high-level transcription might require a BenM tetramer composed of some
subunits bound to benzoate and others bound to muconate . The cartoon
depicts one possible oligomeric configuration for high-level
transcription . However, whether benzoate and muconate bind exclusively
to a subunit remains to be investigated, as does the stoichiometry of
effector binding . Dotted or gray patterns indicate the binding of
muconate or benzoate, respectively, to the BenM protein . Conformational
changes in the protein caused by benzoate may be different from those
caused by muconate, as indicated by different shapes in the
effector-binding regions of the BenM monomers [ovals or rectangles].
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The synergistic effect on transcription may involve some subunits in
the BenM tetramer being bound to benzoate and others being bound to
muconate . The stoichiometry of effector binding within the subunits
and the tetramer remains to be investigated, and the model is meant
solely to represent the idea that a "mixed" oligomer could have a
unique conformation only attainable in the presence of both
compounds . This conformation could be required for the highest-level
transcriptional activation . This regulatory modulation in response to
two effectors provides physiological benefits, since BenM regulates a
complex catabolic pathway in which the accumulation of intermediary
metabolites is toxic . Benzoate initiates increased gene expression,
but maximal transcription would not occur unless muconate signals
that degradation is proceeding appropriately . Gene expression would
be reduced as benzoate availability decreases, yet the signal from
muconate would allow sufficient transcription to complete substrate
consumption . While synergistic response of BenM to two distinct
compounds is novel, further studies are needed to reveal whether
similar mechanisms apply to other transcriptional systems .
This research was supported by National Science Foundation grant
MCB-0212604 to E.L.N .
We thank Juergen Wiegel for helpful discussions .
* Corresponding author . Mailing address: Department of
Microbiology, University of Georgia, Athens, GA 30602-2605 . Phone: [706]
542-2852 . Fax: [706] 542-2674 . E-mail:
eneidle@uga.edu .
Present address: Gastroenterology Research Unit, Mayo Clinic,
Rochester, MN 55905 .
Present address: Center for Tropical and Emerging Global Diseases,
University of Georgia, Athens, GA 30602 .
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