Cloning and characterization of SmeT, a repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF.
We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5' end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PSMET: The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.
Sánchez
Patricia
P
Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain.
Alonso
Ana
A
Martinez
Jose L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D024202:Electrophoretic Mobility Shift Assay; D005798:Genes, Bacterial / Q000235:genetics*; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020615:Stenotrophomonas maltophilia / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012097:Repressor Proteins; D006639:Histidine; D001616:beta-Galactosidase