Previous Article | Next Article 
J Bacteriol, June 1998, p. 2817-2821, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Paradoxical Enhancement of the Activity of a
Bacterial Multidrug Transporter Caused by Substitutions of a
Conserved Residue
Katya A.
Klyachko and
Alexander A.
Neyfakh*
Center for Pharmaceutical Biotechnology and
Department of Medicinal Chemistry and Pharmacognosy, University of
Illinois, Chicago, Illinois 60607
Received 4 February 1998/Accepted 25 March 1998
 |
ABSTRACT |
Substitution of threonine or serine for the evolutionary conserved
intramembrane proline P347 of the Bacillus
subtilis multidrug transporter Bmr significantly increases the
toxin-effluxing activity of Bmr without affecting its abundance in the
cell. In cocultivation experiments, we demonstrate that although the
mutant T347 Bmr is advantageous to cells growing in the
presence of a toxin, the wild-type P347 Bmr is advantageous
under the conditions of nutritional limitation. This may explain why
Bmr has evolved the way it did, that is, with proline at position 347. These observations provide a basis for speculating that the evolution
of Bmr has been determined by its presently unidentified natural
function rather than by its ability to expel diverse toxins from the
cell.
| |
INTRODUCTION |
Bacteria, like eukaryotes, express
multiple membrane transport proteins with unusually low substrate
specificity, so-called multidrug transporters. These proteins, although
not significantly different from their substrate-specific homologs,
recognize, by an unknown mechanism, structurally diverse toxic
compounds and pump them out of cells, thus protecting cells from the
action of toxins (reviewed in references 7 and
8). It is not clear whether the efflux of diverse
toxins is the primary physiological function of multidrug transporters
or, alternatively, is merely a fortuitous side effect of the
unrecognized, more specific functions of these proteins, which may
involve the transport of specific cellular molecules (5).
The subject of this study, the multidrug transporter Bmr of
Bacillus subtilis, promotes the efflux of dissimilar drugs
in exchange for external hydrogen ions (6). On the basis of
sequence homology, Bmr belongs to the evolutionary group of
toxin-extruding antiporter proteins with 12 transmembrane domains
(8, 9). In addition to Bmr, this group includes closely
related bacterial multidrug transporters such as Blt of B. subtilis and NorA of Staphylococcus aureus,
substrate-specific bacterial transporters such as tetracycline efflux
transporters of various bacteria, mammalian monoamine transporters,
whose undisputed function is to transport neurotransmitter molecules
but which are capable of interacting with a variety of toxins
(9), and a large number of uncharacterized eukaryotic and
prokaryotic transporters revealed in the course of genome sequencing
projects (8). These transporters share several conserved
sequence motifs that are presumed to be essential for their function
(8).
Here we demonstrate that the substitution of threonine or serine for
the highly conserved proline residue (P347) located in the
middle of transmembrane domain XI of Bmr increases the toxin-effluxing
activity of this transporter instead of destroying its function, as
could be expected. These results prompted us to question whether the
efflux of toxins has been the determining factor in the evolution of
Bmr.
| |
MATERIALS AND METHODS |
Bacterial strains.
B. subtilis BD170 (thr-5
trpC2) was obtained from the Bacillus Genetics Stock
Center, Ohio State University. Strain BD170-VB, which contains the
bmr gene under the control of the PvegII
promoter, the bmrR gene disrupted by the chloramphenicol
resistance genetic determinant (cat), and the blt
gene disrupted by the erythromycin resistance genetic determinant
(emr), is described in reference 4. The
same reference describes its derivative BD170-VB(V143),
which contains the bmr gene modified to encode the Bmr
transporter with the F143V substitution. Strains
BD170/bmr::cat,
blt::emr, and
BD170/blt::emr are described in
reference 2. Transformation-competent Escherichia coli JM109 was from Promega. Luria-Bertani (LB)
medium was used for cultivating all strains.
Construction of plasmids.
DNA coding for TetC
(tetracycline-specific efflux transporter of class C) was obtained by
PCR using plasmid pBR322 (Sigma) as a template. Substitution of
threonine or serine for P352 was performed by PCR-based
site-directed mutagenesis and verified by direct sequencing of the
entire gene. PCR fragments containing either the wild-type or mutated
promoterless tetC gene were cloned between the
KpnI and EcoRI sites of the plasmid pBluescript
SK+ (Stratagene).
Mutagenesis of B. subtilis.
B. subtilis BD170-VB
or its mutant variant carrying the F143V substitution in Bmr was
mutagenized by ethyl methanesulfonate (Sigma) as previously described
(4) and selected on LB plates containing either 10 µg of
ethidium bromide and 1 µg of reserpine per ml or 15 µg of ethidium
bromide per ml, respectively.
The P347S substitution in strain BD170-VB was created by using
site-directed mutagenesis according to the technique described in
reference 4. For substituting threonine for
P347 in strain BD170, a PCR product containing the mutation
and encompassing the entire bmr and bmrR genes
was generated and used to transform the
BD170/bmr::cat strain. Clones selected
on an LB plate containing 3 µg of ethidium bromide per ml were
analyzed further for the loss of resistance to chloramphenicol. All
substitutions were confirmed by direct local sequencing (ca. 150 bp)
with the fmol PCR-based sequencing system (Promega). Several
clones obtained for each substitution demonstrated indistinguishable
drug resistance characteristics, thus demonstrating that the observed
effects were due to the substitution itself rather than to additional mutations that potentially could occur in the PCR products used for the
mutagenesis.
FLAG modification of Bmr and immunoblotting.
We generated
PCR products which encompassed the entire 5-kb bmr locus of
strain BD170-VB (with PvegII promoter upstream of bmr and cat inserted into the bmrR
gene) or of its Bmr mutants. In addition, a DNA fragment encoding FLAG
epitope (DYKDDDDK) was inserted in frame immediately upstream of the
last three triplets of the bmr gene in these PCR products.
These products were used to transform strain
BD170/blt::erm. Clones were selected
for chloramphenicol resistance (5 µg/ml) and either
tetraphenylphosphonium resistance (50 µg/ml, for the F143V mutant) or
ethidium bromide resistance (10 µg/ml, for all other variants). The
transfer of the mutations and the FLAG epitope-encoding DNA was
confirmed by sequencing.
Cells were grown to an optical density at 600 nm (OD
600) of
0.5, collected by centrifugation, resuspended in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis sample buffer containing
6 M
urea, and loaded on two sodium dodecyl sulfate-12% polyacrylamide
gels. One gel was stained with Coomassie blue; another gel was
analyzed
by immunoblotting after the transfer of proteins to nitrocellulose.
Immunodetection of FLAG-containing proteins was accomplished by
using a
monoclonal anti-FLAG M2 antibody (Kodak) at a 1:1,000
dilution and
horseradish peroxidase-conjugated goat anti-mouse
antibody (Sigma) at a
1:1,000 dilution. To reduce background,
5% dry fat-free milk in
phosphate-buffered saline was used in
all incubations. Peroxidase
activity was detected by the ECL (enhanced
chemiluminescence) Western
blotting detection system (Amersham).
Determination of bacterial sensitivity to drugs.
Cells at a
logarithmic stage of growth (OD600 of 0.4 to 0.8) were
diluted to an OD600 of 0.005 in LB medium. Drugs at 10 different concentrations were added to 1-ml aliquots of cell
suspension. After incubation in a 37°C shaker for 4 to 5 h, at
which point the control culture without added drugs reached
OD600 of approximately 1.0, the optical densities of all
the cultures were measured. The IC50s (50% inhibitory
concentrations) were determined from the obtained
OD600-drug concentration graphs.
Cocultivation experiments.
Strains BD170 and the P347T Bmr
variant of BD170 were grown separately in LB medium to
OD600 of 0.4 to 0.6, diluted to an OD600 of
0.20 in LB medium, and mixed to obtain a 1:1 ratio. Five microliters of
this mixture was added to 5 ml of each of following media: LB, 1%
tryptone medium (1% Difco tryptone, 1% NaCl, 50 µg of threonine per
ml, 50 µg of tryptophan per ml), and 0.1% tryptone medium (0.1%
Difco tryptone, 1% NaCl, 50 µg of threonine per ml, 50 µg of
tryptophan per ml) with or without addition of ethidium bromide (0.2 µg/ml) or reserpine (5 µg/ml). Every 12 h, 5 µl of each
culture was transferred to 5 ml of the corresponding fresh medium.
After 10 such transfers, total DNA from each culture was isolated, and
a PCR product encompassing the bmr gene was sequenced.
| |
RESULTS |
Selection of the P347T and P347S Bmr mutations.
Recently we
described a procedure for selecting mutant variants of Bmr with reduced
sensitivity to its inhibitor, the plant alkaloid reserpine
(4). This procedure uses a specially created B. subtilis strain, BD170-VB, which overexpresses Bmr due to the presence of a strong promoter PvegII inserted immediately
upstream of the chromosomal bmr gene instead of the natural
bmr promoter. Additionally, the gene of the bmr
transcriptional regulator BmrR (1) and the gene of the
second B. subtilis multidrug transporter Blt (2)
are disrupted in this strain. To obtain mutant Bmr variants, BD170-VB
cells are mutagenized with ethyl methanesulfonate and selected on
plates containing a toxic Bmr substrate, e.g., ethidium bromide, in the
presence of reserpine. The absolute majority of the obtained clones
contained amino acid substitutions of Bmr residue F143,
V286, or F306, which led to a dramatic loss of
reserpine sensitivity of Bmr (4).
One of the obtained clones, which has not been described previously,
was unusual in that it demonstrated approximately a twofold
increase in
the level of resistance to dissimilar Bmr substrates,
i.e., ethidium,
norfloxacin, and acriflavine (Table
1),
without
significant changes in the sensitivity of Bmr to inhibition by
reserpine (not shown). Apparently, the increase in the level of
ethidium resistance was sufficient for this clone to survive on
a plate
containing ethidium and reserpine. Sequencing of the entire
bmr gene in this clone revealed a single mutation at the DNA
level
leading to the substitution of threonine for the Bmr residue
P
347 (proline-encoding triplet CCT changed to ACT).
Another substitution of P
347 was subsequently selected in a
different experiment. BD170-VB cells expressing the F143V mutant
variant of Bmr, which provides very little resistance to ethidium,
norfloxacin, and acriflavine (
4), were mutagenized and
selected
for compensatory mutations restoring the ability of the
transporter
to protect cells from ethidium toxicity. As the F143V
substitution
had been artificially created by changing two nucleotides
within
the F
143 codon, the direct reversal to the wild-type
sequence was nearly
impossible. Three of the obtained clones
contained the P347S substitution
(CCT
TCT) in addition to
the F143V mutation. As Table
1 demonstrates,
cells expressing the F143V
P347S variant of Bmr displayed approximately
three- to
five-times-higher levels of resistance to ethidium,
norfloxacin, or
acriflavine than the cells expressing the F143V
variant of Bmr.
Transfer of the P347S Bmr mutation alone into
the BD170-VB cells
resulted in a strain indistinguishable in its
properties from the
strain carrying the P347T mutation of Bmr;
that is, either substitution
of P
347 caused approximately a twofold increase in the
bacterial resistance
to toxic Bmr substrates.
The P347T and P347S substitutions increase the drug transport
activity of Bmr.
The stimulatory effect of the P347T or P347S
substitution on Bmr-mediated drug resistance can be explained either by
the increase in the amount of the Bmr protein in the cell (e.g., due to
its increased half-life) or by the increase in its activity. To discern between these possibilities, we genetically modified the
cytoplasmically located hydrophilic C termini of the wild-type and
mutant Bmr transporters, expressed by the BD170-VB cells. The C termini
of the modified transporters contained the FLAG epitope recognizable by
the M2 monoclonal antibody (3). The FLAG-modified Bmr
variants did not differ from the corresponding unmodified Bmr variants in the ability to provide drug resistance (data not shown).
Figure
1 demonstrates the results of
immunoblotting of the FLAG-modified Bmr variants with M2 monoclonal
antibody. No significant
effects of either the P347T or P347S
substitution on the amounts
of Bmr in the cell could be detected (Fig.
1). This result strongly
suggested that the obtained substitutions of
P
347 stimulate the Bmr-mediated drug resistance by
increasing the
transport activity of Bmr rather than its amount in the
cell.
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 1.
Immunoblotting of FLAG-containing proteins in BD170-VB
B. subtilis cells overexpressing different Bmr variants. Bmr
was either unmodified (lane 1), FLAG modified (lane 2), FLAG-modified
P347T mutant (lane 3), FLAG-modified F143V mutant (lane 4), or
FLAG-modified F143V P347S double mutant (lane 5). Forty micrograms of
total cellular protein was loaded in each lane. The apparent molecular
mass of Bmr is 35 kDa, which is lower than the theoretically predicted
42 kDa, likely due to the high hydrophobicity of this protein. Sizes
are indicated in kilodaltons.
|
|
To verify that these substitutions do indeed increase the drug
transport activity of Bmr, we determined the rates of ethidium
bromide
efflux from BD170-VB cells expressing different FLAG-modified
variants
of Bmr. Cells were loaded with ethidium in the presence
of the Bmr
inhibitor reserpine and then resuspended in a drug-free
medium. The
release of ethidium from the cells was detected by
monitoring the
decline of ethidium fluorescence in the cell suspension
(the
fluorescence of ethidium released from the cells and no longer
associated with nucleic acids or proteins diminishes dramatically).
Figure
2 demonstrates that the P347T
substitution significantly
stimulates the efflux of ethidium. The cells
expressing the F143V
variant of Bmr effluxed ethidium much more slowly
than the cells
expressing wild-type Bmr, which correlated with their
lower resistance.
However, the additional P347S substitution
significantly increased
the ethidium efflux rate (Fig.
2).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 2.
Fluorimetric detection of ethidium efflux from BD170-VB
B. subtilis cells overexpressing different variants of
FLAG-modified Bmr. Cells loaded with ethidium bromide in the presence
of reserpine were placed into drug-free medium, and their fluorescence
( ex = 530 nm; em = 600 nm) was monitored
as described previously (4). Fluorescence is proportional to
the amount of ethidium remaining in the cells. wt, wild type.
|
|
Why has P347 been selected in the course of Bmr
evolution?
The finding that either the P347T or P347S substitution
in Bmr makes it a more efficient multidrug transporter appears
paradoxical from the evolutionary standpoint. Indeed, what is the
reason for proline to be present at position 347 of Bmr if threonine or
serine at this position apparently would make the transporter more
effective?
In model evolution experiments, we demonstrated that the variant of Bmr
with threonine at position 347 does indeed have a
fitness advantage
over the wild-type transporter if the presence
of toxins becomes a
significant factor in the environment of
B. subtilis. A
strain of
B. subtilis which differed from the wild-type
strain BD170 only by the P347T substitution in Bmr was constructed
for
these experiments. As expected, the T
347 strain
demonstrated approximately a two- to three-fold-higher
resistance to
drug substrates of Bmr than the wild-type P
347 strain
(Table
2).
The logarithmically growing P
347 and T
347 cells
were mixed at a ratio of 1:1 and cocultivated for approximately 100 generations.
The ratio of the wild-type and mutant
bmr
sequences in the DNA
prepared from the resulting bacterial population
was then determined.
This ratio remained unchanged if the cocultivation
was performed
in a relatively rich medium, either LB (not shown) or a
medium
containing 1% tryptone and 1% NaCl (Fig.
3A, lane 2). However,
the mutant
T
347 cells completely dominated (Fig.
3A, lane 3) if the
cocultivation
was performed in the presence of as little as 0.2 µg of
the toxic
Bmr substrate ethidium bromide per ml (5% of the MIC of
ethidium
for the wild-type cells).
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 3.
Relative fitness of wild-type B. subtilis
cells expressing either the wild-type P347 or the mutant
T347 variant of Bmr. The cells were mixed at a 1:1 ratio
and then cocultivated in different media for approximately 100 generations. DNA, isolated from the original mixture of cells or from
the cells which underwent cocultivation, was used as a template to
amplify the bmr gene by PCR. Shown are the sequences of the
resulting PCR products (in each lane pair, cytosine at left and adenine
at right). The position of the nucleotide that is different in the two
strains is indicated by an arrow. As shown schematically, cytosine at
this position corresponds to the wild-type sequence (triplet CCT
encoding proline), whereas adenine at this position corresponds to the
mutated sequence (triplet ACT encoding threonine). Results of two
independent experiments are shown in panels A and B. (A) Lane 1, original mixture; lane 2, cocultivation in a nutrient-rich medium (1%
tryptone, 1% NaCl); lane 3, cocultivation in the same rich medium
containing the toxic Bmr substrate ethidium bromide (0.2 µg/ml); lane
4, cocultivation in a poor medium (0.1% tryptone, 1% NaCl). (B) Lane
1, original mixture; lane 2, cocultivation in a poor medium (0.1%
tryptone, 1% NaCl); lane 3, cocultivation in the same poor medium
containing reserpine (5 µg/ml).
|
|
Since normally Bmr contains P
347, one would assume that
under certain environmental conditions this variant should have fitness
advantage over the T
347 variant. In fact, we found this to
be the case in conditions
of a limited supply of nutrients. In a poor
medium (0.1% tryptone,
1% NaCl), the wild-type P
347 cells
repeatedly outgrew the mutant T
347 cells (Fig.
3A, lane 4, and Fig.
3B, lane 2). Importantly, selection
of the wild-type
P
347 variant over the mutant T
347 variant
depended on the function of Bmr. If cocultivation in
a poor medium was
performed in the presence of the Bmr transport
inhibitor reserpine,
neither of the strains gained selective advantage
(Fig.
3B, lane 3).
The P352 substitutions in TetC equivalent to the
P347 substitutions in Bmr.
Proline at position 347 of
Bmr is highly conserved among its homologs and is included into the
conserved motif G (GXXXGP) in the middle of transmembrane domain XI
(8). One of these homologs is TetC, which is encoded by the
E. coli plasmid pBR322. We genetically substituted either
threonine or serine for P352 of TetC, which is equivalent
to P347 of Bmr. The promoterless wild-type and the mutated
tetC genes were then recloned into the pBluescript plasmid
vector under the control of the lac promoter. As expected,
the E. coli cells harboring the plasmid containing the
wild-type tetC demonstrated resistance to tetracycline,
which was dependent on the presence of
isopropyl-1-
-D-galactoside (IPTG), the inducer of the
lac promoter (tetracycline IC50s = 7.5 and
50 µg/ml in the absence and presence of IPTG, respectively). In
contrast, cells harboring the empty vector, or vector carrying either
the P352T or P352S mutant tetC, showed no tetracycline resistance (IC50 = 1 µg/ml regardless of the presence of
IPTG). This result demonstrates that at least some transporters similar to Bmr require the presence of proline in the middle of transmembrane domain XI to be functional. It also underscores the unusual character of our finding that a substitution of this conserved proline in Bmr
leads to an increase in its toxin-effluxing activity.
| |
DISCUSSION |
Our results demonstrate that the P347T or P347S substitution in
the Bmr molecule makes it a more potent multidrug transporter. The
similar properties of the T347 and S347 Bmr
variants are not surprising considering the structural similarity of
threonine and serine. The observed increase in the transporter activity, although easily detectable in both drug resistance and ethidium efflux experiments, is not particularly strong: approximately two- to threefold if the substitution occurs in the wild-type Bmr and
three- to fivefold if it occurs in the transporter weakened by the
F143V mutation. Experiments with the FLAG-modified Bmr variants
indicate that this increase in Bmr activity results not from the
increase in the abundance of Bmr but, most likely, from the increase in
the activity of each individual transporter molecule.
Although the effect of the P347 substitutions is relatively
modest, we were able to find only one publication describing a similar finding: a twofold increase in the transport affinity of lactose permease as a result of the S67A substitution (12). It is
additionally unusual that a substitution improving transporter function
affects an intramembrane proline residue. Prolines in transmembrane
domains of membrane proteins are thought to be of particular importance since they induce kinks in the transmembrane 
-helices and provide the basis for conformational transitions due to cis-trans
isomerization (15). Unlike the substitution described here,
the previously described substitutions of intramembrane prolines were
either detrimental to the function of membrane proteins or, at best, neutral (10, 11, 14, 15).
The evolutionary aspect of the finding described here is the most
paradoxical. Indeed, why would evolution have preserved a residue which
makes a transporter somewhat less efficient than it can potentially be?
As our model evolution experiments demonstrate, the presence of
threonine, instead of proline, at position 347 of Bmr would have
benefited bacteria growing in the presence of toxic Bmr substrates,
even if the toxins were present in the bacterial environment at a
relatively small concentration. Other results of these model
experiments explain, however, why Bmr has evolved the way it did, that
is, with proline being present at position 347. Under the conditions of
nutritional limitation, which are likely to be common for B. subtilis in nature, the P347 variant of Bmr is
superior to the T347 variant. Evidently, in the course of
the evolution of Bmr, this selective pressure has outweighed the
selective pressure imposed by environmental toxins.
One mechanistic explanation of this result postulates that due to its
low specificity, Bmr promotes the efflux of not only toxins but also of
some natural cellular molecules. If the transporter is too active, this
may become detrimental for the cells experiencing nutritional
limitation. In other words, according to this hypothesis, the role of
proline at position 347 of Bmr is to limit the activity of the
transporter and prevent it from becoming damaging to the cell.
This hypothesis can hardly explain, however, another our finding. Since
P347 is conserved in evolution, this proline residue should
be expected to play a similar activity-limiting role in homologous
transporters. Contrary to this prediction, we found that in at least
one of the Bmr homologs, the tetracycline transporter TetC, the proline corresponding to P347 of Bmr (P352) is
absolutely required for the transporter function. To account for this
observation, we would like to propose an alternative explanation for
the results presented here. This alternative hypothesis postulates that
similar to TetC, which requires proline at position 352 to be
functional, Bmr requires proline at position 347 to perform its
natural, presently unidentified specific transport function, which aids
cells experiencing nutritional limitation. The Bmr-mediated efflux of
toxins, according to this hypothesis, is merely a fortuitous side
effect of the true function of Bmr and therefore has never been a
determining factor in the evolution of this transporter. This
hypothesis explains all of the results presented here and is in
agreement with recent findings that the mammalian multidrug transporter
P-glycoprotein may have evolved to transport cellular phospholipids
(13) and that the Bmr homolog, multidrug transporter Blt of
B. subtilis, may have evolved to promote the outward
transport of spermidine (16). We fully realize, however,
that this hypothesis will remain purely speculative until the
hypothetical specific natural function of Bmr is identified.
| |
ACKNOWLEDGMENTS |
We are grateful to A. S. Mankin and P. N. Markham for
helpful discussions and critical reading of the manuscript.
This work was supported by NIH grant GM49819.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Pharmaceutical Biotechnology (M/C 870), University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607. Phone: (312) 996-7231. Fax: (312)
413-9303. E-mail: neyfakh{at}uic.edu.
| |
REFERENCES |
| 1.
|
Ahmed, M.,
C. M. Borsch,
S. S. Taylor,
N. Vazquez-Laslop, and A. A. Neyfakh.
1994.
A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates.
J. Biol. Chem.
269:28506-28513[Abstract/Free Full Text].
|
| 2.
|
Ahmed, M.,
L. Lyass,
P. N. Markham,
S. S. Taylor,
N. Vazquez-Laslop, and A. A. Neyfakh.
1995.
Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated.
J. Bacteriol.
177:3904-3910[Abstract/Free Full Text].
|
| 3.
|
Hopp, T. P.,
K. S. Prickett,
V. Price,
R. T. Libby,
C. J. March,
P. Ceretti,
D. L. Urdal, and P. J. Conlon.
1988.
A short polypeptide marker sequence useful for recombinant protein identification and purification.
Bio/Technology
6:1205-1210.
|
| 4.
|
Klyachko, K. A.,
S. Schuldiner, and A. A. Neyfakh.
1997.
Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr.
J. Bacteriol.
179:2189-2193[Abstract/Free Full Text].
|
| 5.
|
Neyfakh, A. A.
1997.
Natural functions of bacterial multidrug transporters.
Trends Microbiol.
5:309-313[Medline].
|
| 6.
|
Neyfakh, A. A.,
V. E. Bidnenko, and L. B. Chen.
1991.
Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.
Proc. Natl. Acad. Sci. USA
88:4781-4785[Abstract/Free Full Text].
|
| 7.
|
Nikaido, H.
1996.
Multidrug efflux pumps of gram-negative bacteria.
J. Bacteriol.
178:5853-5859[Free Full Text].
|
| 8.
|
Paulsen, I. T.,
M. H. Brown, and R. A. Skurray.
1996.
Proton-dependent multidrug efflux systems.
Microbiol. Rev.
60:575-608[Abstract/Free Full Text].
|
| 9.
|
Schuldiner, S.,
A. Shirvan, and M. Linial.
1995.
Vesicular neurotransmitter transporters: from bacteria to humans.
Physiol. Rev.
75:369-392[Free Full Text].
|
| 10.
|
Sheppard, D. N.,
S. M. Travis,
H. Ishihara, and M. J. Welsh.
1996.
Contribution of proline residues in the membrane-spanning domains of cystic fibrosis transmembrane conductance regulator to chloride channel function.
J. Biol. Chem.
271:14995-15001[Abstract/Free Full Text].
|
| 11.
|
Tamori, Y.,
M. Hashiramoto,
A. E. Clark,
H. Mori,
A. Muraoka,
T. Kadowaki,
G. D. Holman, and M. Kasuga.
1994.
Substitution at Pro385 of GLUT1 perturbs the glucose transport function by reducing conformational flexibility.
J. Biol. Chem.
269:2982-2986[Abstract/Free Full Text].
|
| 12.
|
Tsen, S. D.,
S. C. Lai,
C. P. Pang,
J. I. Lee, and T. H. Wilson.
1996.
Chemostat selection of an Escherichia coli mutant containing permease with enhanced lactose affinity.
Biochem. Biophys. Res. Commun.
224:351-357[Medline].
|
| 13.
|
van Helvoort, A.,
A. J. Smith,
H. Sprong,
I. Fritzsche,
A. H. Schinkel,
P. Borst, and G. van Meer.
1996.
MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine.
Cell
87:507-517[Medline].
|
| 14.
|
Wellner, M.,
I. Monden,
M. M. Mueckler, and K. Keller.
1995.
Functional consequences of proline mutations in the putative transmembrane segments 6 and 10 of the glucose transporter GLUT1.
Eur. J. Biochem.
227:454-458[Medline].
|
| 15.
|
Williams, K. A., and C. M. Deber.
1991.
Proline residues in transmembrane helices: structural or dynamic role?
Biochemistry
30:8919-8923[Medline].
|
| 16.
|
Woolridge, D. P.,
N. Vazquez-Laslop,
P. N. Markham,
M. S. Chevalier,
E. W. Gerner, and A. A. Neyfakh.
1997.
Efflux of the natural polyamine spermidine facilitated by the Bacillus subtilis multidrug transporter Blt.
J. Biol. Chem.
272:8864-8866[Abstract/Free Full Text].
|
J Bacteriol, June 1998, p. 2817-2821, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Garcia, Ma. X. U., Roberts, C., Alexander, H., Stewart, A. M., Harwood, A., Alexander, S., Insall, R. H.
(2002). Methanol and acriflavine resistance in Dictyostelium are caused by loss of catalase. Microbiology
148: 333-340
[Abstract]
[Full Text]
-
Putman, M., van Veen, H. W., Konings, W. N.
(2000). Molecular Properties of Bacterial Multidrug Transporters. Microbiol. Mol. Biol. Rev.
64: 672-693
[Abstract]
[Full Text]
-
Aínsa, J. A., Blokpoel, M. C. J., Otal, I., Young, D. B., De Smet, K. A. L., Martín, C.
(1998). Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present in Mycobacterium fortuitum and Mycobacterium tuberculosis. J. Bacteriol.
180: 5836-5843
[Abstract]
[Full Text]
Top
Abstract
Introduction
