Functional characterization of the Escherichia coli membrane transport protein MdtEF

Abstract

The mdtE and mdtF genes encode membrane transporters, with their transport substrates and mechanisms still not fully understood. Amino acid sequence analysis suggests that MdtE belongs to the MFP family while MdtF belongs to the RND family. MdtF is positioned in the inner membrane, while MdtE is found in the periplasmic space, connecting MdtF with the outer membrane porin TolC to create a MdtEF-TolC tripartite efflux pump for drug and substrate efflux. Subsequently, an Escherichia coli (E. coli) mdtEF gene deletion strain was developed using a suicide plasmid method. An expression vector, was then constructed and complemented based on the pBR322 vector. Drug sensitivity tests and EB accumulation experiments were performed on the mdtEF gene deletion strain, revealing that the MdtEF protein could efflux the carbapenems imipenem and ertapenem. In the EB accumulation experiment, two inhibitors, vanadate and carbonyl m-chlorophenylhydrazone (CCCP), were utilized, demonstrating that MdtEF functions through the proton gradient's potential energy for substrate transport.