Cloning and Characterization of SmeT, a Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

Abstract

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.

Vitamin K 3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump

“…A second treatment was performed using Turbo DNA-free (Ambion). RNA integrity was verified on a 1% agarose gel, and the absence of DNA was confirmed by PCR using primers sme27 and sme48, which amplify a 347-bp fragment belonging to the smeT gene from strain D457 (58). cDNA was obtained from 10 g of RNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems).…”

Stenotrophomonas maltophilia: an Emerging Global Opportunistic Pathogen

“…SmeT was suggested to bind to an operator sequence in the intergenic sequence of smeT-smeD containing the promoters for smeT and smeDEF (301). The overexpression of smeDEF in MDR S. maltophilia strains that harbor wild-type smeT and SmeT binding sites appeared to be influenced by additional mechanisms other than SmeT (302).…”

“…Upstream of the smeDEF operon is smeT, a proposed transcriptional repressor of the smeDEF operon in S. maltophilia (301). SmeT was suggested to bind to an operator sequence in the intergenic sequence of smeT-smeD containing the promoters for smeT and smeDEF (301).…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…For instance, mutations in the regulatory proteins AdeR and AdeL from the opportunistic pathogen A. baumannii result in increased antibiotic resistance due to the overexpression of the AdeABC and AdeFGH pumps, respectively (42,91). Other regulators include AcrR from E. coli and S. enterica, a repressor of the AcrAB pump, and SmeT from Stenotrophomonas maltophilia, which represses the expression of SmeDEF (219). This tight regulation also exists in Gram-positive bacteria.…”