Purification and characterisation of the Escherichia coli multidrug efflux protein MdtM

Multidrug resistance (MDR) is the simultaneous acquisition of resistance by bacteria to a broad spectrum of structurally dissimilar compounds to which they have not been previously exposed. The principle cause of MDR has been identified as the efflux of antimicrobials from the cell by proteins which are integral membrane transporters. Escherichia coli MDR transporter, MdtM, is a previously uncharacterised, 410 amino acid residues protein from the ubiquitous major facilitator superfamily (MFS). This thesis focuses on the purification and characterisation of MdtM. Functional characterisation of MdtM using growth inhibition assays and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to a range of antimicrobial compounds. Functional assays also demonstrated that MdtM functions in alkaline phi homeostasis in E. coli.Inverted vesicle transport assays illustrate the antiport activity of MdtM.<br/><br/>Site-directed mutagenesis studies of MdtM suggest that the residue D22 and the highly conserved residue R108 each play a role in proton recognition. MdtM was homologously overexpressed from a multicopy plasmid and purified to homogeneity in dodecyl-P-maltopyranoside (DDM) detergent solution. The oligomeric state and homogeneity of MdtM in a panel of detergent solutions was analysed using SE-HPLC; MdtM is stable and monomeric in DDM solution. Fluorescence based assays determined the apparent binding affinity of purified MdtM in DDM solution for its substrates.<br/><br/>This thesis indicates MdtM is an exceptionally versatile transporter, with a broad substrate range. This work provides a firm foundation for further biochemical and structural studies of this class of multidrug transporter protein.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

Queen's University Belfast

Year dc:date.issued

2013

Author dc:creator

• Knipe, Scarlett

Language dc:language

eng

Identifier

oai:pure.qub.ac.uk/portal:studenttheses/3f3b97da-c82c-41ad-886f-11212b213f85

OAI identifier oai:identifier

oai:pure.qub.ac.uk/portal:studenttheses/3f3b97da-c82c-41ad-886f-11212b213f85