Characterization of the multidrug efflux regulator AcrR from Escherichia coli

Abstract

The Escherichia coli AcrR represses transcription of the acrB gene, which encodes the multidrug efflux pump AcrB that extrudes a wide variety of toxic compounds, by binding its target operator DNA. Fluorescence polarization was performed using purified, recombinant AcrR that contains a 6xHis tag at the C-terminus and a fluorescein-labeled 28-base pair oligonucleotide bearing a predicted palindrome (IR) operator sequence. Binding of AcrR to the predicted IR sequence occurred with a dissociation constant (K D ) in the nanomolar range. Fluorescence polarization assays were also applied to characterize the affinity and specificity of AcrR interaction with three different fluorescent ligands, rhodamine 6G, ethidium and proflavin. The K D values for these ligands range from 4.2 to 10.1 μM, suggesting that AcrR is capable of recognizing a wide range of structurally dissimilar toxic compounds as it is in the case of the AcrB multidrug efflux pump. We found that the binding of rhodamine 6G to AcrR is inhibited by the presence of ethidium. In contrast, the dissociation constant of proflavin binding to AcrR was not affected by ethidium, a result suggesting that ethidium and proflavin are bound to distinct binding sites.

Crystal structures of CmeR‐bile acid complexes from Campylobacter jejuni

“…8). Such a phenomenon has been previously observed with the crystal structure of QacR simultaneously bound to two ligands, proflavin and ethidium, 19 and has been predicted through biochemical analysis to occur in many other proteins, including AcrR, 22,23 TtgV 24 and MdfA. 25 It has also been reported that the QacR repressor can bind a single ligand in multiple positions, 26 possibly due to the multifaceted nature of this protein.…”

Structural Basis for the Transcriptional Regulation of Heme Homeostasis in Lactococcus lactis

“…Although these TFRs bind different ligands (many structurally unrelated compounds such as ethidium, proflavin, and rhodamine 6G for AcrR (36), saturated fatty acid-CoA and unsaturated fatty acid-CoA for DesT (39,40), and numerous structurally dissimilar monovalent and bivalent cationic and lipophilic compounds for QacR (41,42)), their ligand-binding cavities are similarly positioned in the ligand-binding domain (28). Ligand binding results in a conformational change of the DNA-binding domain, for which the relocation of ␣6 helix upon ligand binding induces a pendulum-like motion of the DNA-binding domain in TFRs including TetR (43), QacR (41), and DesT (39).…”

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

“…Many genes are under either local or global transcriptional control. For example, the multidrug transporter AcrAB is locally controlled by the transcriptional repressor AcrR in Escherichia coli (10,11) and Salmonella enterica (12) and by global transcriptional activators of the AraC/XylS family, such as MarA, SoxS, and Rob (13)(14)(15).…”