Functional gene cloning and characterization of MdeA, a multidrug efflux pump from Staphylococcus aureus.
A DNA fragment conferring drug resistance was cloned from the chromosomal DNA of Staphylococcus aureus N315 using a drug hypersensitive Escherichia coli KAM32 as the host. Although E. coli KAM32 cells were sensitive to many antimicrobial agents, transformed cells harboring a recombinant plasmid carrying the DNA region became resistant to several structurally unrelated antimicrobial agents, such as tetraphenylphosphonium chloride, Hoechst 33342 and norfloxacin. These results suggest that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. We partially determined the nucleotide sequence of the cloned DNA and found the mdeA gene within it. The E. coli cells transformed with the mdeA gene showed efflux activity of Hoechst 33342. On the other hand, S. aureus cells transformed with mdeA showed elevated resistance to doxorubicin, daunorubicin, tetraphenylphosphonium chloride, Hoechst 33342, ethidium bromide and rhodamine 6G. Elevated energy-dependent efflux of ethidium was observed with transformed S. aureus. We found that the mdeA gene was expressed under normal growth conditions in S. aureus N315.
Yamada
Youichi
Y
Department of Molecular Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Japan.
Shiota
Sumiko
S
Mizushima
Tohru
T
Kuroda
Teruo
T
Tsuchiya
Tomofusa
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D001562:Benzimidazoles / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012235:Rhodamines; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001562:Benzimidazoles; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; C510398:MdeA protein, Staphylococcus aureus; D026901:Membrane Transport Proteins; D012235:Rhodamines; C026188:rhodamine 6G; D004996:Ethidium; C017807:bisbenzimide ethoxide trihydrochloride