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 down-regulated 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.

FIGURE 1.

Structure of SmeT. a, ribbon representation of SmeT. The helices 1–9 are displayed in different colors and have been labeled only in subunit A. Subunit A has been superposed onto subunit B and is shown in gray. b, stereo view of the structural comparison between SmeT (green), QacR (magenta, ID code 1JUP) and TtgR (red, ID code 2UXU). The figure shows the extended N and C termini of SmeT. c, conservation of the hydrophobic character of the residues in the interface of SmeT dimer. The figure has been prepared using HOMOLMAPPER based on an alignment of 288 sequences from members of the TetR family prepared by M-Coffee. Residues in green have a conserved hydrophobic character, and residues in magenta do not. Subunit B has been represented as a yellow backbone tube to show the dimerization area. d, electrostatic surface potential of SmeT dimer. Because the model is incomplete, subunit A was superposed onto subunit B, and the dimer generated in that way was used for the calculations. The electrostatic potential was calculated by APBS and is mapped on the solvent-accessible surface by CHIMERA. Electrostatic potential values range from positive (blue, 5 kT/e) to negative (red, -5 kT/e), where k is Boltzmann's constant, T is the absolute temperature, and e is the proton charge.

FIGURE 2.

Effector binding pocket of SmeT. a, the figure shows the secondary structure of subunit A of SmeT in pale brown, and the binding pocket is represented in magenta. Pathways leading to this cavity were calculated using CAVER and are represented using PYMOL. b, the surface of SmeT subunit A is in green, and subunit B is in blue, showing the entrance between helices 4, 5, and 6. The binding pocket for subunit A is represented in magenta. c, stereo view of the residues lining the hydrophobic cavity of SmeT.

FIGURE 3.

Binding of SmeT to its operator DNA. a, identification of the SmeT binding site by a DNase I-footprinting assay. An end-labeled 223-bp DNA segment containing the smeD-smeT intergenic region was incubated with increasing concentrations of SmeT and later digested by DNase I. SmeT protects in both DNA strands a 28-bp sequence located between positions -14 and -41 from the smeD transcription start site. Two inverted repeats located within the protected region (IR1 and IR2, indicated by arrows) may represent the binding sequences for SmeT. IR1p, inverted repeat 1 proximal arm (relative to the dyad that relates the two dimers); IR1d, inverted repeat 1 distal arm; IR2p, inverted repeat 2 proximal arm; IR2d, inverted repeat 2 distal arm. smeD and smeT transcription start sites are indicated with a box. The -35 and -10 regions of both promoters are highlighted in bold. b, binding to a 30-bp DNA that contains the putative SmeT operator. The end-labeled 30-bp DNA was incubated with increasing concentrations (0, 0.2, 0.4, 0.8, 1.6, and 3.2 μm) of SmeT (lanes 1–6, respectively). Complexes were resolved in a 6% (w/v) nondenaturating polyacrylamide gel. With SmeT at 3.2 μm, the binding was competed by the unlabeled probe (1 and 10 μm, lanes 7 and 8. c, critical nucleosides for SmeT binding to DNA. Missing nucleoside assays were performed with the whole end-labeled 223-bp intergenic region for the smeD strand and a 158-bp fragment containing the hypothetical SmeT binding site for the smeT strand. Both DNA fragments were subjected to hydroxyl radical cleavage and were later incubated with SmeT. Bound and free DNA were separated on a 6% (w/v) polyacrylamide nondenaturing gel, purified from the gel, and analyzed in 8% (w/v) urea-polyacrylamide gels. Nucleosides critical for SmeT binding in both strands are indicated with bars. M, DNA sequence ladder; F, free DNA; B, bound DNA. d, SmeT-Operator DNA binding model. SmeT binds to a 28-bp region placed between positions -14 and -41 from the smeD transcriptional start site. This operator DNA contains a pseudopalindromic sequence with two overlapping inverted repeats (IR1 and IR2) that can accommodate a pair of SmeT dimers each. Our results suggest that the strongest interaction is carried out by the SmeT dimer that binds to the IR1 in the smeD coding strand through binding to the sequence TGTATGT. Once this first dimer is bound, the second dimer binds cooperatively. Critical nucleosides for SmeT binding are underlined. smeD and smeT transcription start sites are indicated with a box.