Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli.
Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of Neisseria gonorrhoeae and the YdhE protein of Escherichia coli have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro. E. coli strains expressing norM or ydhE showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in E. coli, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na(+)-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region.
Long
Feng
F
Molecular, Cellular, and Developmental Biology Interdepartmental Graduate Program, Iowa State University, Ames, Iowa 50011, USA.
Rouquette-Loughlin
Corinne
C
Shafer
William M
WM
Yu
Edward W
EW
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism / Q000494:pharmacology; D005454:Fluorescence Polarization; D008826:Microbial Sensitivity Tests; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009643:Norfloxacin / Q000378:metabolism / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D017920:Antiporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C114619:NorM protein, bacteria; D004996:Ethidium; D009643:Norfloxacin