The role of MfsR, a TetR-type transcriptional regulator, in adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride via the regulation of mfsQ

Abstract

A gene encoding the TetR-type transcriptional regulator mfsR is located immediately downstream of mfsQ and is transcribed in the same transcriptional unit. mfsQ encodes a major facilitator superfamily (MFS) efflux transporter contributing to the resistance of Stenotrophomonas maltophilia towards disinfectants belonging to quaternary ammonium compounds (QACs), which include benzalkonium chloride (BAC). Phylogenetic analysis revealed that MfsR is closely related to CgmR, a QAC-responsive transcriptional regulator belonging to the TetR family. MfsR regulated the expression of the mfsQR operon in a QAC-inducible manner. The constitutively high transcript level of mfsQ in an mfsR mutant indicated that MfsR functions as a transcriptional repressor of the mfsQR operon. Electrophoretic mobility shift assays showed that purified MfsR specifically bound to the putative promoter region of mfsQR, and in vitro treatments with QACs led to the release of MfsR from binding complexes. DNase I protection assays revealed that the MfsR binding box comprises inverted palindromic sequences located between motifs -35 and -10 of the putative mfsQR promoter. BAC-induced adaptive protection was abolished in the mfsR mutant and was restored in the complemented mutant. Overall, MfsR is a QACs-sensing regulator that controls the expression of mfsQ. In the absence of QACs, MfsR binds to the box located in the mfsQR promoter and represses its transcription. The presence of QACs derepresses MfsR activity, allowing RNA polymerase binding and transcription of mfsQR. This MfsR-MsfQ system enables S. maltophilia to withstand high levels of QACs.

Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens

“…We identified seven TA systems in A. faecis and three in A. lanthieri (Table 5 ). The Tet R-type transcriptional regulator is located near a gene encoding a major facilitator superfamily (MFS) efflux transporter (Table 5 ), showing the resistance to disinfectants of quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) [ 64 ]. Also, hip BA TA systems are present in both species (Table 5 ).…”

Multi-species biofilms of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride

“…which had an MIC of 25 ppm. Stenotrophomonas maltophilia , an opportunistic human pathogen, can carry an efflux transporter involved in decreased susceptibility to BAC (i.e., msfQ ) [ 59 ], however, this gene was not detected in any Xanthomonadaceae isolates included in this study. Other genes related to quaternary ammonium resistance were detected in some Xanthomonadaceae isolates, including qacA , emrE , and mexAB genes.…”

Contribution of Stenotrophomonas maltophilia MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitR

“…The DNase I protection assay was performed as previously described [ 23 ]. Briefly, the binding reaction was set in a 20-μL reaction mixture containing 50 ng [ 32 P]-labeled 204-bp mfsBC promoter fragments prepared as described for EMSA and increasing concentrations of purified DitR (0–50 nM).…”

“…To purify DitR, an exponential-phase culture of E. coli BL21 (DE3)/pLacI harboring pET-DitR was induced with 250 μM isopropyl-β-D-thiogalactopyranoside overnight at 25˚C. The 6×His-tag DitR protein was purified as previously described [23] using a column containing nickel-nitrilotriacetic acid resin (Qiagen, France) and elute with 150 mM imidazole. The purity of purified DitR was estimated by SDS-PAGE and densitometric analysis of the Coomassie blue-stained gels (S1 Fig) . Fractions with DitR purity greater than 95% were used in further experiments.…”