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J Biol Chem, Vol. 275, Issue 9, 6101-6106, March 3, 2000
From the Center for Adaptation Genetics and Drug Resistance and
Department of Molecular Biology and Microbiology, Tufts University
School of Medicine, Boston, Massachusetts 02111
An interdomain hybrid Tet protein consisting of a
class C Tetracycline (Tc)1
resistance is mediated by many different but related determinants in
Gram-negative bacteria, designated as classes A through E, G, H, and J
(1). Each determinant encodes a cytoplasmic membrane protein, Tet, that
catalyzes the active efflux of a tetracycline-divalent cation complex
from the cell in exchange for a proton (2). Tet proteins are members of
the major facilitator superfamily that includes transporters of various drugs, antibiotics, sugars, and the well-studied lactose permease (3).
Members of this group share a common topology as well as regions of
amino acid sequence identity.
The Tet proteins of classes B and C are predicted to contain 12 transmembrane (TM) regions divided in half into two domains, Tc resistance proteins from classes A and C share 78% amino acid
sequence identity, whereas the class B determinant is more distantly
related to the other two, exhibiting 45% identity (15, 16). A hybrid
Tet protein consisting of a class A In the present study, we have isolated and characterized active
Tet(C/B) mutants. The locations of the specific mutations identify
areas that probably mediate functional interactions between the Reagents--
Restriction enzymes were obtained from New England
Biolabs (Beverly, MA). Taq polymerase and T4 DNA ligase were
purchased from Life Technologies, Inc. Antibiotics were obtained from
Sigma, except that AHTc was prepared by Mark Nelson of this laboratory. [3H]Tc was purchased from NEN Life Science Products. The
remaining reagents were analytical grade.
Bacterial Strains and Plasmids--
Escherichia coli
DH5
Plasmid pRAR1033 carrying a tet(C/B) hybrid gene (17) was
used in both hydroxylamine and PCR mutagenesis and for expression of
the wild-type and mutated Tet(C/B) proteins. Plasmid pLR1068 served as
a source of Tet(B/B) (class B Random Mutagenesis of Plasmid DNA and Isolation of Tc-resistant
Mutants--
Plasmid DNA was purified using a Qiaprep spin miniprep
kit (Qiagen, Valencia, CA). 3 µg of plasmid pRAR1033 DNA was
incubated in 400 mM hydroxylamine, 50 mM
potassium phosphate (pH 6.6), 100 mM EDTA in a 100-µl
volume for 1 h at 65 °C followed by overnight incubation at
37 °C. The entire volume was drop-dialyzed against sterile distilled
water for 2 h at room temperature on a type VS filter (0.025 µM pore size; Millipore, Bedford, MA). After ethanol
precipitation and a 70% ethanol wash, the DNA was suspended in sterile
distilled water.
E. coli DH5
Specific mutations within the tet(C/B) gene were identified
by DNA sequencing. The entire coding region of hybrid tet
genes containing mutations in either domain was subcloned on a
Xmn I/BglII fragment into the original
unmutagenized plasmid (pRAR1033) to ensure that any Tc resistance
observed was due solely to the identified mutation.
The mutations that produced Tc resistance by Tet(C/B) were introduced
into the wild-type Tet protein class from which they were derived. The
G247D mutation was introduced into the class B tet gene on
pLR1068 by replacement of an EcoRI/PvuI fragment. A BamHI/SalI fragment of pRAR1033 encoding the
G152E mutation was exchanged into the tet(C) gene on pCR2.
Using the same restriction sites, the G152E mutation was also added to
a tet(C/B) hybrid gene already containing the G247D mutation
to create a double mutant. The exchanges were verified by sequencing.
Site-directed Mutagenesis--
Site-directed mutagenesis of the
(C/B) hybrid tet gene on plasmid pRAR1033 or the class C
tet gene was performed by a PCR overlap method (22).
Mutagenic PCR primers were designed to incorporate a restriction
endonuclease site along with the desired mutation where possible (Table
I). The final PCR product potentially carrying the specific mutation was digested with PvuI and
BamHI and exchanged into the unmutated parental
tet gene. Plasmids were screened for the specific mutation
by restriction enzyme analysis. The integrity of the mutated
tet genes was verified by DNA sequencing.
DNA Sequencing--
Sequencing of plasmid DNA was performed at
the Tufts University Core Facility using a 373 stretch DNA sequencer
(Applied Biosystems).
Tc Susceptibility--
Cells harboring various wild-type and
hybrid tet genes were grown in the presence of
chloramphenicol and AHTc to an A530 of 0.8. Cells were swabbed for confluent growth onto an LB plate containing
AHTc. E-test strips (a gift of AB Biodisk, Solna, Sweden) containing Tc
were applied to the inoculated plate. After overnight incubation at
37 °C, growth inhibition around the test strip corresponding to the
MIC of Tc was examined.
Membrane Isolation and Western Blot Analysis--
DH5
Before electrophoresis, membrane fractions were incubated in reducing
sample buffer (21) for 1 h at 37 °C. Proteins were separated on
a 10% polyacrylamide gel, then electroblotted to an Immobilon-P
membrane (Millipore) as per the manufacturer's directions. The blot
was probed with a polyclonal antibody specific for the 14 carboxyl-terminal amino acid residues of Tet(B) (anti-Ct antibody,
kindly provided by A. Yamaguchi (24)) followed by a secondary antibody
conjugated to horseradish peroxidase (Phototope-HRP; New England
Biolabs). Blots were developed with a Renaissance Western blot
chemiluminescence kit (NEN Life Science Products).
Detection of Tet(C) Proteins--
To detect Tet(C) proteins with
the anti-Ct antibody, the 6 carboxyl-terminal amino acids were removed
and replaced with the 14 terminal amino acids of Tet(B). This was
achieved by inserting a HpaI site between base pairs 1169 and 1170 of tet(C) by PCR amplification. A
BamHI/HpaI fragment encompassing all but the last
22 base pairs of the class B gene was then excised from plasmid pRAR1033 and replaced with the corresponding fragment of
tet(C). The resulting construct encoded Tet(C), with the
anti-Ct epitope replacing the carboxyl-terminal cytoplasmic tail. This
procedure was also used to alter the Tet(C) S202F and G152E mutants.
Tc Efflux Assays--
Assays were performed with E. coli AG100A cells as the chromosomal deletion in the
acr locus eliminated host background Acr-mediated efflux of
Tc. Energy-dependent Tc efflux was measured as the amount of [3H]Tc taken up by cells expressing various Tet
proteins in the energized versus deenergized states. Assays
were performed essentially as described by McMurry et al.
(25), with the exception that cells were grown in liquid LB medium
containing 0.2% glucose in the presence of AHTc to induce expression
of Tet protein. Time points began upon the addition of 4 µM [3H]Tc. CCCP was added to a final
concentration of 100 µM at 20 min to deenergize the
cells. Each strain was assayed in triplicate.
Generation of Tc-resistant Mutants--
A total of 21 Tc-resistant
mutants were independently isolated from separate mutagenic and
transformation events. Sequence analysis of the hybrid tet
gene from each mutant revealed three types of mutations, each
consisting of a single base change (Table II).
A GGA
In nine additional mutants, a GGC
Finally, three of the Tet(C/B) mutants isolated contained a UCC to UUC
codon change that resulted in a Ser-202
Each type of mutation was subsequently introduced into the Tet protein
from which the corresponding region of the Tet(C/B) protein was
derived. Introduction of the G152E mutation into Tet(C/C) (consisting
of both an Expression of Parental Versus Mutant Hybrid Tet
Proteins--
Western blot analysis of the proteins was performed
using the anti-Ct antibody directed against the 14 carboxyl-terminal
amino acids of Tet(B). The
The parental hybrid Tet(C/B) protein was abundantly present in the
membranes of the cells in which it was expressed (Fig. 2A, lane 3). The
near background level of Tc susceptibility of Tet(C/B) has been
attributed to inefficient functional interactions between
The G152E and S202F C/B proteins were detected in the membrane in
quantities comparable with the original inactive Tet(C/B) hybrid (Fig.
2A, lanes 3, 4, and 11).
The G247D mutant was detected in slightly lower quantities in the
membrane compared with the original Tet(C/B) and the other mutant
proteins (Fig. 2A, lane 9). Although combining
the G152E and G247D mutations in Tet(C/B) did not produce resistance to
Tc, the protein was observed in higher quantities in the membrane than
the original hybrid or any of the single mutants (Fig. 2B,
lane 5).
A strain expressing Tet(B) protein containing the G247D mutation was
inactive, which could be attributed to a decreased amount of this
mutant protein in the membrane compared with the wild type (Fig.
2B, lane 3). The similar lack of Tc resistance by
the Tet(C) G152E could also be due, at least in part, to lower protein expression (Fig. 2C, lane 3). This protein also
had a higher mobility in the gel relative to the wild type, which may
reflect either a conformational change or the charge difference between
the two proteins. It is unlikely, however, to be due to truncation of the G152E protein, as recognition by the anti-Ct antibody requires an
intact carboxyl terminus. In addition, a Tet(C) protein containing the
G152E mutation complements a deficiency in potassium transport (26), an
activity that requires the amino terminus of Tet(C) to be associated
with the membrane (27). The presence of the S202F mutation in Tet(C)
(generated by site-directed mutagenesis) did not affect the amount of
protein in the membrane (Fig. 2C, lane 4). In
general, although the Tet(C) proteins analyzed by Western blot were
engineered to express the anti-Ct epitope at their carboxyl termini,
even the wild-type protein was not as reactive with the antibody as
Tet(B) (compare Fig. 2C, lanes 2 and
5). In addition, although Tet(C) with the anti-Ct tag
contained only two more amino acid residues, it had a noticeably lower
mobility than Tet(B) (Fig. 2C).
Site-directed Mutants--
It was of interest to determine whether
the negatively charged side chain at either 152 or 247 was a requisite
for Tc resistance by the hybrid protein. To this end, G152Q and G247N
mutants were generated by site-directed mutagenesis. The Tet(C/B) G247N
mutant provided a moderate level of Tc resistance (24 µg/ml; Table
III). This protein was observed in the membrane in a similar quantity to the wild-type Tet(C/B) hybrid (Fig. 2A, lane
10) and more than the G247D mutant (Fig. 2A, lane
9). Cells expressing the G152Q mutant, however, failed to show the
Tet(C/B) protein in the membrane (Fig. 2A, lane
7) and were susceptible to Tc (Table III).
To further investigate the side chain requirements at position 152, additional replacements were constructed. Ala, Asp, and Lys were each
substituted for Gly-152; only G152D resulted in a protein capable of
mediating a significant level of Tc resistance (10 µg/ml; Table III).
Each mutant protein was detectable in the membrane (Fig.
2A), although the G152K protein was present in relatively
lower amounts.
These results indicated that an acidic residue was required at position
152 to produce active Tet(C/B). Furthermore, introduction of a neutral
or positively charged side chain at position 152 decreased the
stability of the Tet(C/B) protein. In contrast, replacing Gly-247 with
either Asp or Asn was structurally tolerated and also rendered Tet(C/B)
capable of mediating Tc resistance.
Tc Efflux Assays--
Energy-dependent Tc efflux was
measured as the relative uptake of [3H]Tc before and
after deenergization of the cells with the protonophore CCCP. As shown
in Fig. 3, AG100A cells not expressing a
Tet protein accumulated 26 pmol of [3H]Tc/mg of total
protein at 20 min. The addition of 100 µM CCCP resulted
in decreased accumulation of Tc in the cell, attributed to the
dissipation of the proton gradient across the membrane upon which Tc
uptake is dependent. The wild-type class B protein expressed from
pLR1068 demonstrated a relatively low uptake of [3H]Tc (9 pmol/mg of protein at 20 min). Deenergization of the cells by CCCP
resulted in an increased accumulation of [3H]Tc due to
elimination of active Tc efflux. Cells expressing the unmutated
Tet(C/B) hybrid from plasmid pRAR1033 did not exhibit efflux
activity.
The G152E and G247N Tet(C/B) mutants carried out Tc efflux as well as
Tet(B) or Tet(C) (data not shown), accumulating only 8.5 and 9 pmol of
[3H]Tc/mg of protein, respectively, at 20 min. Efflux by
the G247D and S202F mutants resulted in an accumulation of
approximately 11 pmol of [3H]Tc/mg of protein at 20 min.
Overall, resistance to Tc mediated by each of the mutant Tet(C/B)
proteins was accompanied by energy-dependent efflux of Tc
from the cell, although as noted before (8), Tc efflux activity did not
directly correlate with the level of Tc resistance.
Three different single amino acid changes out of a total of 21 independently isolated mutants restored Tc resistance to an otherwise
inactive hybrid Tet(C/B) protein. Each mutant demonstrated a MIC for Tc
above the 10 µg/ml on which they were selected. Of the 5 × 105 transformants screened, nine contained a G152E mutation
in helix 5 of the class C Both Gly-152 and Gly-247 are conserved among all classes of
Gram-negative Tet proteins and are predicted to be relatively close to
the periplasmic side of the membrane (Fig. 1). As conserved amino acid
residues are usually of functional or structural significance, it was
expected that substitutions leading to an active Tet(C/B) would have
been at nonconserved positions. Substitution of Gly-152 with Glu in
Tet(C) or Gly-247 with Asp or Asn in Tet(B) had a detrimental effect on
Tc resistance, indicating the importance of Gly at these two positions
in the wild-type proteins.
Gly-152 is the last residue of an amino acid sequence motif
(GX8GX3GPX2GG)
found among the antiporter members of the major facilitator superfamily
(28). Our finding that a G152E mutation inactivates Tet(C) agrees with
the findings of McNicholas et al. (26). Substitutions at
other positions within this conserved motif in Tet(C), specifically
Gly-147 (replaced with any other amino acid) (28) or Gly-143 (replaced
with Asp or Asn) (26), also reduced or eliminated Tc resistance. Based
on molecular modeling of helix 5 of Tet(C), Gly residues within this
sequence were concluded to participate in the formation of a substrate
binding pocket devoid of side chains (28). If this model holds true,
then our result implies that within the Tet(C/B) hybrid, the putative
Tc binding site is ineffective, and a G152E mutation suppresses this defect. The fact that only a negatively charged side chain at position
152 could render Tet(C/B) active suggests that an ionic interaction is
formed with another region of the protein or that the side chain
contributes in some manner to Tc binding. Residues within helix 5 are
important for substrate recognition by other major facilitator
superfamily members, particularly the multidrug transporter
Bmr (29) and the lactose permease (30).
With respect to Gly-247, a helical wheel projection predicts that it is
located on the same hydrophilic side of amphipathic helix 8 as Gln-261
and His-257. Gln-261 appears to contribute to substrate recognition by
Tet(B) (31), and His-257 has been proposed to play an essential role in
proton translocation (13). Introduction of the G247D mutation into
Tet(B) apparently destabilized the protein, as this protein was present
in reduced quantities in membrane vesicles relative to the wild type.
In Tet(C/B), substitution of Gly-247 with a larger, hydrophilic (Asp or
Asn) residue may also perturb the structure of the helix, at least
enough to render the protein active without severely displacing the key
amino acid side chains at positions 257 and 261. If Gly-247 does indeed
face, or is proximal to, the Tc/proton transport pathway rather than the membrane, introduction of the hydrophilic side chains of Asp or Asn
at that position would not necessarily be thermodynamically unfavorable.
Although the presence of either G152E or G247D is sufficient to
activate Tet(C/B), the addition of the second mutation inactivates the
protein without decreasing its stability (Fig. 2B,
lane 5). Considering that the class C and class B proteins
share a relatively low sequence similarity (45%) (15, 16), it is
surprising that a single amino acid substitution could render the
previously inactive Tet(C/B) hybrid functional. In this sense, the
individual mutations act as suppressors that compensate for the large
differences in the amino acid sequences between the respective domains
of the two classes, whereas the presence of the second mutation
"desuppresses" the compensatory action of the first. This effect
supports the hypothesis that a specific functional interaction exists
between regions of helices 5 and 8. Alternatively, the mutations could reflect two entirely independent alterations, which individually promote functional interactions between Not much attention has been given to the interdomain loop of Tet
proteins owing to their poor amino acid sequence conservation. However,
the random isolation of the active Tet(C/B) S202F mutant, without any
additional mutations in the membrane-spanning regions, demonstrates
that the interdomain loop is functionally important. Ser-202 is a
nonconserved residue, found only in Tet classes C and H. The fact that
the S202F replacement inactivates Tet(C) indicates that Ser-202 somehow
contributes to Tc resistance. This finding confirms and extends other
studies, which suggest that other residues within the interdomain
region are important for Tet function (4, 33, 34).
Overall, our results strongly indicate that helices 5 and 8 interact
and provide further evidence that the interdomain loop has a role in
the function of Tet proteins.
We thank Laura McMurry and Michael N. Alekshun for helpful discussions and critical reading of this manuscript.
*
This work was supported by National Institutes of Health
Grant GM55430.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.
The abbreviations used are:
Tc, tetracycline;
TM
transmembrane domain, AHTc, 5
Evidence for Interactions between Helices 5 and 8 and a Role
for the Interdomain Loop in Tetracycline Resistance Mediated by
Hybrid Tet Proteins*
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MATERIALS AND METHODS
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domain and a class B 
domain (Tet(C/B)) lacks detectable
efflux ability and provides only minimal levels of resistance to
tetracycline (Tc) (3 µg/ml) compared with intact class B (256 µg/ml) and class C (64 µg/ml). Twenty-one independently isolated
mutants of the Tet(C/B) protein with increased Tc resistance were
generated by random chemical mutagenesis. Nine mutants with a Glu
substitution for Gly-152 in helix 5 of the class C domain produced
a resistance of 48 µg/ml, whereas another 9 with an Asp replacement
of Gly-247 in helix 8 of the class B domain mediated resistance at
32 µg/ml. The third type of mutation, found in 3 mutants expressing
24 µg/ml resistance, was a S202F replacement in the putative
interdomain cytoplasmic loop of Tet(C/B). The latter underscores a
previously unappreciated function of the interdomain cytoplasmic loop.
All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored
energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could
also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively
charged side chain at position 152 was required for Tet(C/B) activity.
These mutations appear to promote the necessary functional interactions
between the interclass domains that do not occur in the Tet(C/B) hybrid
protein and suggest a direct association between helix 5 and helix 8 in
the function of Tet efflux proteins.
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and
, by a large putative cytoplasmic loop designated the interdomain region (4-7). Extensive genetic and mutagenic analyses of the class B
Tet protein has shown that the two domains, both of which are required
for Tet function (8), contribute differently to the efflux of Tc. In
the domain, amino acid residues in TM2 and TM3 line a putative
substrate translocation pathway (9, 10). The cytoplasmic loop
connecting TM2 and TM3 could act as a gate that undergoes a
conformational change during Tc transport (11, 12). In the domain,
His-257 in TM8 appears to be involved in proton translocation (13),
whereas Asp-285 in TM9 is important for Tc binding (14).
domain and class C domain
(Tet(A/C)) specified levels of Tc resistance not unexpected for the
combination of the two functional proteins (17). In contrast, a
Tet(C/B) protein (class C domain and class B domain) provided
only minimal resistance to Tc, and a Tet(B/C) protein (class B domain and class C domain) did not confer resistance (17). However,
an easily detectable level of Tc resistance (10-15% that of the level
of wild-type Tet(C)) was observed when both the Tet(C/B) and the
Tet(B/C) proteins were expressed together in the same cell. This result
indicated that the individual domains of each hybrid protein were
active and capable of interacting with the corresponding opposite
domain of the same class (18). Presumably, differences between the amino acid sequences of the class C and class B prevented the domains
of the Tet(C/B) hybrid from functioning.
and
domains of Tet proteins and also implicate the interdomain region
in the function of the Tet protein.
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cells (recA1; laboratory collection) were used for
the propagation of plasmid DNA, determining Tc resistance, and for
assessing protein expression. Tc efflux assays were performed with
E. coli AG100A cells (
acrAB (19)). Cell
cultures were routinely grown in LB media supplemented with
chloramphenicol (20 µg/ml) or Tc (10 µg/ml), as needed. AHTc (0.025 µg/ml) was used as a gratuitous inducer of Tet protein where applicable.
domain and class B domain) (20).
A XhoI/BglII fragment of pRAR1013, carrying the tet(C) gene (17), was used to replace the
tet(C/B) hybrid gene in pRAR1033. The resulting construct,
pCR2, was used as a source of Tet(C/C) (class C domain and class C
domain) in this study.
cells were transformed with 50 ng of
mutagenized DNA using the CaCl2 heat shock method (21).
Upon recovery, a total of 5 × 105 transformants were
plated on LB plates containing 20 µg/ml chloramphenicol (for
maintenance of the plasmid) and 10 µg/ml Tc. Apparent Tc-resistant mutants were visible after 24-48 h of incubation at 37 °C. Only one
colony per transformation was chosen for analysis to ensure that the
mutants isolated were generated in separate mutagenic events. Tc
resistance was verified by overnight growth of liquid LB cultures
containing chloramphenicol and Tc. Plasmid DNA was isolated from
Tc-resistant cultures and retransformed, and transformants were grown
on Tc to verify that resistance was plasmid-mediated.
Site-directed mutants created in this study
cells
expressing various Tet proteins were grown in the presence of AHTc, and
membranes were prepared from them as described previously (23).
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Effect of specific mutations in Tet(C/B) hybrids on the minimum
inhibitory concentration (MIC) of Tc
GAA transition within codon 152 resulted in a Gly to Glu
mutation in nine different mutants (Table II). Gly-152 is located in
putative helix 5 of the domain of Tet(C) in the Tet(C/B) hybrid
(Fig. 1). The Tc MIC of cells expressing
this protein was 48 µg/ml, approximately 16-fold greater than that of
the original Tet(C/B) protein (3 µg/ml). This Gly residue (position
152 in Tet(C)) is conserved among all Tet proteins in Gram-negative
bacteria.
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Fig. 1.
Predicted topology of the C/B hybrid Tet
proteins, based on the respective topological models of the class B
(35) and class C (34) Tet proteins. Circled letters
indicate the residues that were substituted in Tc-resistant Tet(C/B)
mutants; Gly-152 in helix 5 of Tet(C) and Gly-247 in helix 8 of Tet(B)
protein are shown. Ser-202 is in the interdomain cytoplasmic loop and
the class C half of the hybrid protein. The junction between class C
and class B is indicated by an arrow.
GAC transition resulted in the
substitution of Gly-247 of the class B domain in Tet(C/B) with an
Asp residue (Table II). Based on the current topological model of
Tet(B), the location of Gly-247 is predicted to be within putative
helix 8 of the domain of the Tet(C/B) protein (Fig. 1). Gly at
position 247 of Tet(B) is also conserved in all Gram-negative Tet
proteins. Cells expressing the G247D Tet(C/B) mutant protein had a Tc
MIC of 24 µg/ml, approximately 8-fold over the original Tet(C/B) protein.
Phe mutation. A host
bearing this mutation exhibited a Tc MIC of 32 µg/ml. Ser-202 is
within the Tet(C) domain of the Tet(C/B) hybrid and resides in the
interdomain cytoplasmic loop connecting the and domains (Fig.
1).
and a domain from the class C protein) or the G247D
mutation into Tet(B/B) (comprised of class B and domains)
resulted in inactive proteins, as demonstrated by a Tc MIC of 1.5 µg/ml. A S202F mutation in Tet(C) also led to a severe reduction in
Tc resistance to 4 µg/ml from 64 µg/ml mediated by the wild-type protein.
domain of Tet(C) proteins was altered so that they would be detected by anti-Ct (see "Materials and
Methods"). This replacement resulted in a small increase in the Tc
MIC provided by these proteins (Table
III).
Tc resistance mediated by wild-type, hybrid, and mutant hybrid Tet
proteins
and domains from the two different classes (17).
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Fig. 2.
Western blot of wild-type, hybrid, and mutant
hybrid Tet proteins. Membrane fractions were prepared from DH5
cells expressing various Tet proteins, and 2.5 µg of total membrane
protein per lane was loaded, with the exception that
membrane containing Tet(C) proteins were loaded at 10 µg/lane. Blotted proteins were probed with an antibody
specific for the 14 carboxyl-terminal amino acids of Tet(B).
A, lanes 1, no Tet protein; 2, Tet
(B/B); 3, Tet(C/B); 4, Tet(C/B) G152E;
5, Tet(C/B) G152A; 6, Tet(C/B) G152D;
7, Tet(C/B) G152Q; 8, Tet(C/B) G152K;
9, Tet(C/B) G247D; 10, Tet(C/B) G247N;
11, Tet(C/B)S202F. B, lanes 1, no Tet
protein; 2, Tet(B/B); 3, Tet(B/B) G247D;
4, Tet(C/B); 5, Tet(C/B) G152E/G247D.
C, lanes 1, no Tet protein; 2,
Tet(C/C) with anti-Ct tag; 3, Tet(C/C) G152E with anti-Ct
tag; 4, Tet(C/C) S202F with anti-Ct tag; 5,
Tet(B/B).
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Fig. 3.
Uptake of [3H]-Tc by E. coli AG100A cells expressing wild-type, hybrid, or mutant
hybrid Tet proteins. Accumulation was measure as pmol of
[3H]Tc accumulated/mg of protein. Assays were performed
at 30 °C with 4 µM [3H]Tc added after a
5-min preincubation in the presence of 0.2% glucose. CCCP (100 µM) addition is indicated by an arrow.
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domain, and nine more contained the G247D
replacement in helix 8 of the class B domain. This finding suggests
that critical interactions between the and the domains that are required for the function of Tet(C/B) occur between helices 5 and 8.
and domains but
together, do not. Our results are consistent with the general model for the arrangement of the TM regions of major facilitator superfamily members proposed by Goswitz and Brooker (32), in which helices 5 and 8 are predicted to be adjacent to one another and line the substrate
translocation channel.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Center for Adaptation
Genetics and Drug Resistance, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6764; Fax:
617-636-0458.
ABBREVIATIONS
,6-anhydrotetracycline;
PCR, polymerase
chain reaction;
MIC, minimum inhibitory concentration;
CCCP, carbonyl
cyanide m-chlorophenylhydrazone;
[3H]Tc, [7-3H(N)]Tc.
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MATERIALS AND METHODS
RESULTS
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REFERENCES
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