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Antimicrobial Agents and Chemotherapy, November 2000, p. 3079-3086, Vol. 44, No. 11
Departamento de Biotecnología
Microbiana, Centro Nacional de Biotecnología, CSIC, Campus
Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain
Received 20 March 2000/Returned for modification 16 July
2000/Accepted 18 August 2000
Stenotrophomonas maltophilia is a nosocomial bacterial
pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous
multidrug-resistant S. maltophilia D457R mutant (A. Alonso
and J. L. Martinez, Antimicrob. Agents Chemother. 41:1140-1142,
1997) was screened for complementation of erythromycin susceptibility
on an antibiotic-hypersusceptible Escherichia coli
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Characterization of SmeDEF, a Novel
Multidrug Efflux Pump from Stenotrophomonas
maltophilia
acrAB
strain. Cloning and further analysis revealed that a 6-kbp region
constituting a transcriptional unit was capable of complementing the
antibiotic-susceptible phenotype of an E. coli
acrAB
strain. We identified three open reading frames, smeD, smeE and smeF, which code for members of the
membrane fusion protein, resistance nodulation division, and outer
membrane factor families, respectively. Drug susceptibility assays
indicated that the SmeDEF system cloned in E. coli mediates
resistance to a wide range of antibiotics. Ethidium bromide and
norfloxacin accumulation experiments in the presence and in the absence
of carbonyl cyanide m-chlorophenylhydrazone showed that
this system constitutes a drug efflux pump dependent on the membrane
proton motive force. The presence of high levels of smeDEF
mRNA in the multiresistant D457R mutant was consistent with the high
levels of SmeF (formerly Omp54) observed in the same strain. In
contrast, transcription levels of smeDEF in the D457 strain
were tiny, which correlates with the low levels of SmeF observed for
this strain. Also, for both the D457 and D457R strains, we observed
growth phase-dependent regulation in which the highest level of
transcription corresponded to early exponential phase, with
transcription decreasing throughout the growth curve to undetectable
levels at 24 h.
*
Corresponding author. Mailing address: Departamento de
Biotecnología Microbiana, Centro Nacional de
Biotecnología, CSIC, Campus Universidad Autónoma de
Madrid, Cantoblanco, 28049-Madrid, Spain. Phone: 34-91-5854551. Fax:
34-91-5854506. E-mail: jmtnez{at}cnb.uam.es.
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