title: Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM.
abstract: Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl-β-D-maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl-β-D-maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, UK.
authors.forename: Scarlett R
authors.initials: SR
authors.lastname: Holdsworth
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Law
mesh_terms: D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; C574623:MdtM protein, E coli; D026901:Membrane Transport Proteins