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Antimicrobial Agents and Chemotherapy, June 2004, p . 2274-2276, Vol . 48, No . 6
Regulatory Regions of smeDEF in Stenotrophomonas maltophilia Strains Expressing Different Amounts of the Multidrug Efflux Pump SmeDEF
Patricia Sanchez, Ana Alonso, and Jose L . Martinez*
Departamento
de Biotecnología Microbiana, Centro Nacional de
Biotecnología, CSIC, Campus Universidad Autónoma
de Madrid, Cantoblanco, 28049-Madrid, Spain
Received 22 July 2003/
Returned for modification 3 November 2003/
Accepted 21 February 2004
The
smeT-smeDEF region and the smeT gene, which encodes
the smeDEF repressor, are highly polymorphic . Few changes in
smeT might be associated with smeDEF overexpression.
The results obtained with cellular extracts suggest that mutant SmeT
proteins cannot bind to the operator and that other transcription
factors besides SmeT are involved in the regulation of smeDEF
expression .
The expression of the Stenotrophomonas maltophilia multidrug
resistance (MDR) pump SmeDEF is transcriptionally regulated by SmeT
(12), a local repressor
encoded by the smeT gene, which is located upstream of
smeD and which is divergently transcribed . SmeT binds to the
intergenic smeT-smeD region, where the promoters of
smeT and smeD are located . It has
previously been found that a mutation in smeT is responsible
for smeDEF overproduction in MDR strain S.
maltophilia D457R (1,
12) . However, nothing is
known about the molecular basis of smeDEF overproduction in
clinical S . maltophilia isolates . For that goal, the
intergenic smeT-smeD region, which contains both the
smeT and smeD promoters, as well as the smeT
gene, were cloned from a collection of clinical S . maltophilia
strains, 33% of which were SmeDEF overproducers
(2), by PCR and sequenced
as described previously
(12) . The strains used in
this work are listed in Table 1 . The intergenic region and the smeT gene were highly
polymorphic (Fig.
1) . However, only some changes might account for smeDEF
overproduction . Most nucleotide changes were located outside of the
region between the transcriptional origins of smeT and
smeD, which suggests that their operator sequences are located
between the smeD-smeT transcriptional origins . Only one
smeDEF-overproducing strain (strain E923) contained
modifications within the smeT-smeD region compared with the
sequence of wild-type strain D457 . The nucleotide changes in E923 were
exactly the same as those in antibiotic-susceptible strain E759 . Thus,
those changes are not associated with the smeDEF-overproducing
phenotype .
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TABLE 1 . Bacterial
strains
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FIG . 1 . Schematic
representation of the intergenic smeD-smeT region
(A) and the smeT gene (B) . MDR strains are marked
with an asterisk . Single base substitutions are represented in black.
Continuous, dashed, and dashed-dotted lines indicate different base
substitutions in a given position . Base insertions are highlighted in
red . Base deletions are highlighted in blue . The diamonds in panel B
represent mutations producing a nonconservative amino acid change that
can be involved in the MDR phenotype (see text and Table 2).
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Most nucleotide changes in smeT did not
render changes in its amino acid sequence . Furthermore, most amino acid
changes in the smeDEF-overproducing strains were also present
in some wild-type strains (Table 2) . Six amino acid changes (underlined in Table 2) were exclusively found
in the MDR strains and thus might be responsible for the MDR phenotype.
Two MDR strains had one change each: clinical isolate E729 had a
Thr197Pro substitution and in vitro mutant D457R had a Leu166Gln
change . Strain E923 had two amino acid changes: the Leu166Gln
substitution observed in D457R and another Arg123Lys change . Finally,
strain C357 had four changes: Arg123Lys (found in E923), Leu144Pro,
Arg148Gln, and Ala204Glu . SmeDEF-nonoverproducing strain E999 contained
a similar substitution at position 148, the change being in this case
Arg148Lys . Only four amino acid changes (boldface in Table 2) were nonconservative:
Leu144Pro, Leu166Gln, Thr197Pro, and Ala204Glu . All of the changes were
clustered in the carboxylic region of the SmeT protein, suggesting a
relevant role of the carboxy terminus of SmeT in its
function .
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TABLE 2 . Amino
acid changes in SmeT proteins of clinical S . maltophilia
isolates
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The sequences of smeT and the intergenic
smeT-smeD region were exactly the same as those of strains
F861 (wild type) and F375 (smeDEF overproducer) . Thus,
smeDEF overproduction in F375 is the consequence of mutations
in other loci, and factors other than SmeT must be involved in the
regulation of smeDEF . A similar situation was described in
nalC Pseudomonas aeruginosa mutants that overexpress the
mexAB-oprM multidrug efflux pump but that do not have
mutations in the mexR gene encoding its local transcriptional
repressor (13).
Increasing evidence supports the idea that the regulation of MDR pump
expression is complex (6).
One of the clearest examples of this is the regulation of
acrAB expression in Escherichia coli, in which
several transcription factors are involved
(10,
11) . So, it seems that
MDR pumps expression needs to be finely tuned, probably in response to
different environmental inputs .
Using whole-cell extracts and a
purified His-tagged SmeT protein, we showed that the wild-type SmeT
protein binds to the intergenic smeT-smeD region, whereas the
Leu166Gln SmeT mutant was incapable of such binding
(12) . Although the
intergenic smeT-smeD region is polymorphic in our clinical
isolates, the region between the transcriptional origins of
smeT and smeD is conserved . Since this region
comprises the operator sequences of smeT and smeD
and, most probably, the binding sites of SmeT, we analyzed wild-type
strain S . maltophilia D457, one of our clinical isolates, for
the presence of cellular factors capable of binding to the intergenic
smeT-smeD region . As shown in Fig.
2, all isolates that did not overproduce smeDEF had a protein(s)
that was able to bind to the promoter region of the operon in the same
way as the wild-type strain D457 does . The retarded complex was of the
same size as that obtained by using a His-tagged recombinant SmeT
protein . On the other hand, the MDR strains with mutations in SmeT
presented the same pattern as D457R, an MDR mutant obtained in the
laboratory . This indicates that mutant SmeT proteins of these strains
are unable to bind to the intergenic smeT-smeD region . We did
find, however, that cellular extracts from MDR strain F375 were able to
bind to the intergenic smeT-smeD region . These data agree with
the fact that SmeT in this strain did not have any relevant amino acid
change . Since in all cases mutations map out of the HTH motif, an
explanation for the lack of SmeT activity might be a reduced stability
of the protein . By using an anti-SmeT antibody obtained in our
laboratory, it has been determined by Western blotting
(3) that the amount of
SmeT was variable among these clinical isolates; however, none of the
MDR mutants presented lower SmeT levels than wild-type strains (data
not shown), indicating that the quantity of SmeT is not the limiting
factor in the MDR phenotype .
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FIG . 2 . Extracts
obtained from wild-type S . maltophilia strains are capable of
retarding the intergenic smeT-smeD region . Two retarding
complexes (marked with arrows) were detected in the case of extracts
from the wild-type strains . These complexes were not detected when
extracts from MDR strains, (marked with an asterisk) were assayed . A
band smaller than that corresponding to the SmeT-DNA complex was
observed in the band shifts by using cellular extracts from the
smeDEF-overproducing mutants . With the available data, it
difficult to know whether this band is nonspecific or whether it is the
consequence of the binding of a cellular factor required for
smeDEF transcription . Cellular extracts from E . coli
M15(pPS6), which contains a His-tagged SmeT protein able to bind to and
retard the intergenic smeT-smeD region
(12), were used as
controls . The band shift obtained with this recombinant SmeT protein is
dose dependent and is of the same size as one of the bands obtained
with extracts from S . maltophilia D457 . 6xHis, six-His
tag.
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Few data comparing sequences from
different S . maltophilia strains are available; however, a
high degree of diversity similar to that observed in this work was
previously reported for beta-lactamases
(5) and the topoisomerase
II and IV quinolone resistance-determining regions
(14) . Bacterial diversity
and evolution can be driven by mutation
(8) and recombination
(7,
9) . Data from our work and
others indicate that mutation might have a relevant role in the
evolution of antibiotic resistance in S . maltophilia . However,
comparison of the DNA sequences comprising smeT and the
intergenic smeT-smeD region in strains E759, E923, and E999
has shown that they have a mosaic structure, with some completely
conserved regions and other divergent regions, with very clear
boundaries between them . Previous work in our laboratory has shown the
presence of genes in the genome of S . maltophilia that
originated from gram-positive organisms
(4) . Together, these data
indicate that recombination should also have a relevant role in the
evolution of S . maltophilia .
We thank Fernando Rojo for fruitful discussions and
suggestions on this work .
The research in our laboratory is aided
by grants QLRT-2000-1339, QLRT-2000-00873, BIO2001-1081,
CAM08.2/0020-1/2001, and
GEN2001/4689/C05 .
* Corresponding
author . Mailing address: Departamento de Biotecnología
Microbiana, Centro Nacional de Biotecnología, CSIC, Campus
Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain.
Phone: 34-91-5854542 . Fax: 34-91-5854506 . E-mail:
jlmtnez{at}cnb.uam.es .
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