Structural and Functional Analysis of SmeT, the Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

Abstract

Stenotrophomonas maltophilia is an opportunistic pathogen characterized for its intrinsic low susceptibility to several antibiotics. Part of this low susceptibility relies on the expression of chromosomally encoded multidrug efflux pumps, with SmeDEF being the most relevant antibiotic resistance efflux pump so far studied in this bacterial species. Expression of smeDEF is downregulated by the SmeT repressor, encoded upstream smeDEF, in its complementary DNA strand. In the present article we present the crystal structure of SmeT and analyze its interactions with its cognate operator. Like other members of the TetR family of transcriptional repressors, SmeT behaves as a dimer and presents some common structural features with other TetR proteins like TtgR, QacR, and TetR. Differing from other TetR proteins for which the structure is available, SmeT turned out to have two extensions at the N and C termini that might be relevant for its function. Besides, SmeT presents the smallest binding pocket so far described in the TetR family of transcriptional repressors, which may correlate with a specific type and range of effectors. In vitro studies revealed that SmeT binds to a 28-bp pseudopalindromic region, forming two complexes. This operator region was found to overlap the promoters of smeT and smeDEF. This finding is consistent with a role for SmeT simultaneously down-regulating smeT and smeDEF transcription, likely by steric hindrance on RNA polymerase binding to DNA.Opportunistic pathogens intrinsically resistant to antibiotics are currently a relevant health problem (1). Although several elements contribute to the intrinsic resistance of these bacteria (2, 3), the active efflux of antibiotics is a common mechanism relevant for their phenotype. Antibiotic efflux is because of the activity of multidrug (MDR) 5 efflux pumps (4 -7). Those elements are universally distributed among all living systems (8). In Gram-negative bacteria, the best characterized MDR pumps belong to the resistance nodulation division family. In most cases MDR pumps are down-regulated by specific transcription factors located upstream of the operon coding for the pump (9). Stable expression and, thus, a higher level of resistance is achieved by mutations in these regulatory elements. It is worth noting here that the constitutive expression of MDR pumps in these mutants is frequently linked to fitness costs (10 -12). This indicates the need of a precise and stringent regulation of the system to avoid the nonspecific expression of these elements.Stenotrophomonas maltophilia is an opportunistic pathogen that is considered as a prototype of intrinsically resistant bacteria (13). The first MDR pump described in this bacterial species was SmeDEF (14 -16), an MDR determinant that contributes to intrinsic (17) and acquired (14, 16) resistance to several antibiotics and to biocides (18) in S. maltophilia. Expression of smeDEF is down-regulated by the transcriptional repressor SmeT (19,20), located upstream, and in the complementary strand of smeDEF. Mut...

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…A Leu166Gln substitution in SmeT results in higher expression levels of both smeDEF and smeT (863). SmeT functions as a dimer, like other TetR proteins (727), but has two extensions at the N and C termini, with a small binding pocket observed in the TetR family repressors (864). SmeDEF overproduction was also found in an isolate with an IS1246-like element in the smeDEF promoter where SmeT likely binds, suggesting that an IS element may also influence smeDEF expression (576).…”

“…SmeABC is positively controlled by SmeSR, as is evident with the reduced MDR due to the smeR deletion (571). Located upstream of the smeDEF operon, smeT encodes the most studied pump repressor SmeT in S. maltophilia (863,864). The promoter regions of smeDEF and smeT are overlapping, and SmeT provides negative control to SmeDEF and its own expression.…”

The TetR Family of Regulators

“…8). For example, ActR, QacR, SmeT, TetR, and TtgR all have a "side entry" opening distal to the dimerization interface that is believed to be the site of access for different ligands (22,(51)(52)(53)(54). Ligands appear to enter CmeR, CgmR, HrtR, LfrR, and SimR via an entry point closer to the "front" of the protein (43,46,50,55,56).…”

A Function of SmeDEF, the Major Quinolone Resistance Determinant of Stenotrophomonas maltophilia, Is the Colonization of Plant Roots

“…The probe for the electrophoretic mobility shift assay (EMSA) was a 158-bp double-stranded DNA fragment containing the SmeT operator site, and 5=-end labeled with [␥-32 P]dATP, which was obtained as previously described (27). This probe was incubated with purified SmeT (0.2 M) in binding buffer [10 mM Tris-HCl, 50 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, pH 7.2, 50 g/ml bovine serum albumin, 1 mM dithiothreitol, 5% (vol/vol) glycerol, and 100 g/ml poly(dI:dC), as a nonspecific competitor DNA] for 20 min at room temperature.…”