Open AccessMutations in M ex B that affect the efflux of antibiotics with cytoplasmic targets
Author(s) -
OheneAgyei Thelma,
Lea Jon D.,
Venter Henrietta
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2012.02594.x
Subject(s) - efflux , periplasmic space , biochemistry , cytoplasm , biology , mutant , atp binding cassette transporter , amino acid , transporter , membrane transport protein , mutation , microbiology and biotechnology , gene , escherichia coli
Drug efflux pumps such as M ex AB ‐ O pr M from P seudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein M ex B is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance–nodulation–division ( RND ) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of M ex B . The two conserved phenylalanine residues have been mutated to alanine residues ( FAFA M ex B ). The interaction of the wild‐type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic‐binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in G ram‐negative pathogens.