Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a.
The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.
Hearn
Elizabeth M
EM
Department of Biological Sciences, CW405 Biological Sciences Center, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
Gray
Murray R
MR
Foght
Julia M
JM
D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019897:Periplasm; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism / Q000494:pharmacology; D011551:Pseudomonas fluorescens / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011084:Polycyclic Aromatic Hydrocarbons; D014050:Toluene