Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Functional Characterization of Putative Escherichia coli ABC Multidrug Efflux Transporter YddA

  • Feng, Zhenyue (College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University) ;
  • Liu, Defu (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Liu, Ziwen (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Liang, Yimin (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Wang, Yanhong (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Liu, Qingpeng (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Liu, Zhenhua (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Zang, Zhongjing (College of Life Science and Technology, Heilongjiang Bayi Agricultural University) ;
  • Cui, Yudong (College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University)
  • Received : 2020.03.31
  • Accepted : 2020.04.23
  • Published : 2020.07.28
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Abstract

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drug-sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

Keywords

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