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Journal of Bacteriology, May 1999, p. 3303-3306, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of MarR Superrepressor
Mutants
Michael N.
Alekshun1,2 and
Stuart B.
Levy1,2,3,*
Center for Adaptation Genetics and Drug
Resistance,1 and Departments of
Molecular Biology and Microbiology2 and of
Medicine,3 Tufts University School of
Medicine, Boston, Massachusetts 02111
Received 14 December 1998/Accepted 21 March 1999
 |
ABSTRACT |
MarR negatively regulates expression of the multiple antibiotic
resistance (mar) locus in Escherichia coli.
Superrepressor mutants, generated in order to study regions of MarR
required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro.
Mutations occurred in three areas of the relatively small MarR protein
(144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.
| |
TEXT |
The chromosomal multiple antibiotic
resistance (mar) locus of Escherichia coli
controls an adaptational response to antibiotics and other
environmental hazards (1). The expression of multiple genes
on the E. coli chromosome is regulated by MarA, a
transcriptional activator encoded within the marRAB
operon (1).
MarR negatively regulates expression of the marRAB
operon (6, 15, 20). DNA footprinting experiments
suggest that MarR dimerizes at two locations, sites I and II, within
the mar operator (marO) (15); site I
is positioned among the 
35 and 10 hexamers, and site II spans the
putative MarR ribosome binding site (see Fig. 1A) (reviewed in
reference 1). Many structurally dissimilar chemicals
affect MarR activity in whole cells (4, 7, 15, 20).
Experiments in vitro demonstrate that MarR binds salicylic acid and,
through the use of gel mobility shift assays, that sodium salicylate
inhibits the formation of MarR-marO complexes
(15). We have extended these initial findings by
demonstrating that the DNA binding activity of MarR in vitro is
antagonized by several other chemicals (2). Thus, MarR
possesses DNA binding and effector molecule recognition properties. In
order to study MarR structure and function, we have generated and
characterized MarR superrepressor (MarRS) mutants.
Bacterial strains, plasmids, and genetic techniques.
The
bacterial strains and plasmids used are listed in Table
1. A low-copy-number wild-type MarR
expression vector was constructed by using a modified version of pACT7
(14). marR was amplified by PCR from E. coli AG100 (8) chromosomal DNA by using Taq DNA polymerase in accordance with the manufacturer's protocols (Life
Technologies, Gaithersburg, Md.). EcoRI and PstI
restriction sites were incorporated into the forward and reverse
primers to facilitate directional cloning into pACT7 in place of the T7
RNA polymerase gene following digestion with EcoRI and
PstI. In pAC-MarR (WT), transcription of marR is
regulated by the lacP1 promoter and protein
synthesis is governed by the wild-type MarR ribosome binding site
(AGGG) and translational initiation (GTG) signals (6).
A high-copy-number wild-type MarR expression vector was
constructed in pET13a (22), a kanamycin-resistant
version of pET11a (Novagen, Madison, Wis.). PCR amplification of
marR was performed as described above by using forward and
reverse primers containing VspI and BamHI
restriction sites to facilitate directional cloning into
NdeI/BamHI-digested pET13a. In the
resulting plasmid, pMarR-WT, expression of MarR is under the control of
the T7 RNA polymerase promoter and a near-consensus ribosome binding site.
For functional analysis of MarR in whole cells, a
PmarII-marO::ccdB
fusion was created in pET11d (Novagen). After digestion with
EcoRV to remove the majority of lacI, pET11d was
purified with the Qiagen (Santa Clarita, Calif.) gel purification kit
and religated. PmarII-marO,
containing the marRAB promoter
(PmarII) and operator (marO)
sequences (Fig. 1A), was amplified by PCR with primers containing EagI/BsmI restriction
sites and blunt-end cloned into
EagI/BsmI-digested pET11d (lacking
lacI), creating plasmid pmarO. Subsequently, the
lacO-ccdB portion of pKIL 18 (5) was amplified by
PCR to exclude the tac promoter sequences. XhoI
and BsmI restriction sites were incorporated into the
forward and reverse primers to facilitate directional cloning
downstream of PmarII-marO, into
AvaI-digested (mixed cohesive and blunt ends) pmarO. The resulting plasmid was designated pSup-Test (Fig.
1B).
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FIG. 1.
(A) Sequence of
PmarII-marO. The locations of the
35 and 10 hexamer sequences and MarR ribosome binding site are
indicated, and the SspI restriction enzyme recognition
sequence in site I is in boldface. (B) Map of plasmid pSup-Test.
Expression of ccdB is positively controlled by the
marRAB promoter (PmarII) and
negatively regulated by the lac (lacO) and
marRAB (marO) operators in the presence of their
cognate proteins, LacI and MarR. The positions of the SspI
sites within the plasmid are indicated.
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DNA sequence analysis was performed (in-house) with an ABI automated
DNA sequencer. Hydroxylamine and nitrosoguanidine mutagenesis of
pAC-MarR (WT) were performed in accordance with established protocols
(17).
Selection of MarR superrepressors.
To identify MarR
superrepressors, expression of the lethal ccdB gene product
on pSup-Test was exploited. Plasmid pAC-MarR (WT) was mutagenized in
vitro and transformed into DH5
containing pSup-Test. Transformants
were selected in the presence of sodium salicylate, a known
marRAB operon inducer (7). Growth of
DH5 bearing pET11d or pmarO (pSup-Test lacking
ccdB) (Table 1) was unaffected by the highest concentration
of this and other inducers tested (Table
2). DH5 cells containing pSup-Test in
the absence or presence of plasmid-encoded wild-type marR
were nonviable in the presence of sodium salicylate and other inducers
(Table 2). However, cells containing a putative MarR superrepressor
survived higher concentrations of known marRAB
operon inducers, presumably by binding of the mutant protein to
marO in front of ccdB on pSup-Test and preventing
expression of the lethal gene product (Table 2). From a total of 276 transformants, 12 putative MarR superrepressor mutants were
independently isolated (Fig. 2).
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TABLE 2.
Survival of DH5 containing pSup-Test and a plasmid
bearing wild-type MarR or putative superrepressor MarR mutant on
gradient plates containing inducing compounds
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FIG. 2.
Locations of the MarR superrepressor mutations
identified in this study. The designations in parentheses consist of
the single-letter code for the wild-type residue followed by the
location of that amino acid in the full-length MarR and the
single-letter code for the mutation isolated at this point. The numbers
in parentheses represent the number of independent isolates at that
site. The cross-hatched box indicates the region of amino acid homology
among the MarR family members.
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|
Assay of repressor activity by 
-galactosidase.
The
trans-dominant nature of the mutant plasmids was then
independently retested in E. coli SPC105, a
marR+ host which contains a chromosomally
located PmarII::lacZ fusion at
the 
attachment site (7).
In the absence of exogenously provided wild-type MarR, SPC105 exhibited
an easily detectable basal level of LacZ expression (Fig.
3). LacZ expression in cells bearing
wild-type marR in trans was minimal and was
virtually undetectable in cells containing any of the five putative
MarRS mutants (Fig. 3). Sodium salicylate caused a
15-fold increase in LacZ expression in cells bearing plasmid-encoded
wild-type MarR, while those containing a MarRS mutant
displayed greatly reduced responses to this inducer (Fig. 3). The G95S,
D26N, D26N/R27H, and V132M mutants showed little if any response, while
cells containing the L135F MarRS mutation showed partial
responsiveness to the inducer (Fig. 3).
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FIG. 3.
MarR repressor activity assayed in the
PmarII::lacZ reporter strain
E. coli SPC105 (marR+). Cells were
grown at 37°C to mid-logarithmic phase in LB broth, without glucose,
containing the appropriate antibiotics and IPTG (50 µM) and sodium
salicylate (5 mM) where appropriate. -Galactosidase assays were
performed with cells permeabilized with chloroform-SDS as previously
described (17, 20). The relative -galactosidase
activities (±standard deviations) in the absence (open bars) or
presence (hatched bars) of 5 mM sodium salicylate are presented.
Activities were determined for the host strain alone (None), the
wild-type MarR (WT), and other MarR proteins that show a
superrepressor phenotype (mutations are indicated below each bar).
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|
Properties of MarRS mutants.
DH5 bearing the
D26N, D26N/R27H, G95S, or V132M MarR mutant proteins displayed similar
decreased susceptibilities to the chemically induced expression of the
lethal ccdB gene product as assayed by gradient plates
(Table 2). The inducer responsiveness of the L135F MarR
mutant-containing cells was less than that of the wild-type control
cells but greater than that of cells bearing the other superrepressor
mutants (Table 2).
Western blot analysis using MarR polyclonal antibodies, generated in
rabbits by using purified MarR (Covance Research Products Inc., Denver,
Pa.), showed that cells bearing the D26N, D26N/R27H, and L135F mutants
expressed 1.3- to 2.0-fold more protein than did those with the
wild-type MarR (data not shown). The intracellular levels of the G95S
and V132M mutant proteins were less, 20 and 40%, respectively, of
wild-type MarR (data not shown). These results demonstrated that
superrepression was not likely attributable to overexpression of the
mutant proteins. This point was addressed more clearly by an in vitro
DNA binding assay.
The wild-type and marR superrepressor genes were cloned into
pET13a and transformed into E. coli BL21(DE3) for
overexpression, and the MarR proteins were purified. Cells, grown in
Luria-Bertani (LB) broth at 37°C to mid-logarithmic phase, were
induced for 3 h with 1 mM IPTG
(isopropyl--D-thiogalactopyranoside), collected, washed,
and frozen at 70°C. The frozen cell pellet was resuspended in 10 ml
of buffer A [50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 2.5 mM
4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF; serine
protease inhibitor) (Sigma, St. Louis, Mo.)], and lysed by sonication.
After removal of insoluble matter by centrifugation at 30,000 × g for 1 h, the supernatant was loaded onto a
sulphopropyl (SP)-Sepharose HiTrap column (Pharmacia Biotech,
Piscataway, N.J.) equilibrated with 50 mM Tris-HCl (pH 7.4). Following
a 50 mM Tris-HCl (pH 7.4) wash, MarR was eluted with a linear gradient
of 0 to 1 M NaCl in 50 mM Tris-HCl (pH 7.4). Eluting at 0.2 to 0.3 mM NaCl, MarR was dialyzed against 100 volumes of 50 mM Tris-HCl (pH
7.4)-100 mM NaCl-10% glycerol-1 mM phenylmethylsulfonyl fluoride (serine protease inhibitor) overnight at 4°C. Judged to be >90% pure on a sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis Coomassie blue-stained gel, MarR was stored in aliquots
at 70°C until further use.
A unique SspI recognition sequence within one of two MarR
binding sites in marO (Fig. 1A) formed the basis of a
restriction enzyme site protection assay (10, 16, 21) to
assess MarR binding to marO. Serial dilutions of purified
wild-type or MarR superrepressor proteins were added to a final volume
of 20 µl containing 0.2 µg of pSup-Test (target DNA, 3.4 nM), 10 mM
Tris-HCl (pH 7.5), 5 mM NaCl, 1 mM MgCl2, and 0.0025%
Triton X-100. After incubation at room temperature for 10 min, 5 U of
SspI (New England Biolabs, Beverly, Mass.) was added and the
reaction mixture was incubated for 30 min at 37°C. The incubation was
terminated by the addition of 1.5 µl of stop buffer (0.25M EDTA [pH
8.0], 1% SDS) and 5 µl of 6× agarose gel loading buffer (0.25%
bromophenol blue, 0.25% xylene cyanol, 30% glycerol) and analyzed on
0.7% agarose (Life Technologies) gels. The point of 50% protection, determined by visual inspection of ethidium bromide-stained DNA in
these gels, was assigned a value of 5 U of activity. The specific activity was then calculated from this value as previously described (10).
The D26N, G95S, and L135F superrepressor mutant proteins displayed at
least a ~9-fold-greater DNA binding activity than the wild-type
repressor (Table 3). Although the inducer
susceptibility profiles of these three mutants were similar in intact
E. coli DH5 (Fig. 3 and Table 2), their in vitro DNA
binding properties were quite different (Table 3). The DNA binding
level of the G95S MarRS mutant was ~2- and 3.5-fold
greater than that of the D26N and L135F mutants, respectively (Table
3).
The G95S MarRS mutation occurred within a region that is
conserved among all members of the MarR family of proteins (Fig.
2), and the mutant protein is 30-fold more active than wild-type MarR. trans-dominant negative complementing MarR mutants
that are in proximity to this residue (3, 6, 20) suggest
that it may play a more direct role in DNA binding.
The D26N superrepressor mutation results in a charged amino acid being
substituted for an uncharged residue. A decrease in electrostatic
interactions between the protein and the DNA backbone may be the basis
for this superrepressor activity. It is also possible that new hydrogen
bonds between the asparagine side chain and the DNA backbone contribute
to an increased affinity for DNA. Thus, nonspecific DNA binding is
expected to form the basis of superrepression. In the D26N/R27H double
mutant, the latter mutation is probably not required since this mutant
produced data similar to that of the protein bearing the single D26N
change (Fig. 3 and Table 2).
The V132M MarRS mutant showed inducer responses like those
of the D26N and G95S mutants in whole cells (Table 2). Since both mutations are expected to lie outside of the putative DNA binding domain of MarR (3), it is speculated that each plays an
accessory role in DNA binding. With respect to the L135F mutant, the
phenylalanine residue may increase DNA binding through newly acquired
interactions with the phosphate backbone (19). The lesion in
each mutant may also reside in a region required for proper protein
folding, MarR oligomer assembly, or an inducer recognition domain. It
is also possible that the superrepressor mutation in these or the other
proteins affects transmission of the signal to the DNA binding domain
following inducer recognition.
That the MarR superrepressor mutations are scattered throughout the
protein suggests that amino acid changes in several regions (Fig. 2)
can enhance the DNA binding activity of the repressor (Table 3). Four
interspersed missense mutations in TrpR resulted in superrepressor
proteins (9, 11, 12), and none displayed altered binding
affinities for the corepressor tryptophan (9). Whether
enhanced DNA binding is attributable to an increased association or
decreased dissociation rate of repressor-operator complex formation or
altered DNA complex stoichiometry, as was demonstrated for particular
TrpR superrepressors (9, 13), is currently unknown. It is of
interest that only one of the TrpR superrepressor mutations lay within
the protein's helix-turn-helix DNA binding domain (18). This finding demonstrates that there is no a priori reason to suspect
that a mutation must be confined to the DNA binding domain of MarR in
order to result in a superrepressor phenotype.
| |
ACKNOWLEDGMENTS |
We are grateful to Philippe Bernard for supplying the pKIL vectors.
This work was supported by NIH grant GM 51661.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Tufts University
School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6764. Fax: (617) 636-0458. E-mail:
slevy{at}opal.tufts.edu.
| |
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Journal of Bacteriology, May 1999, p. 3303-3306, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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