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. 2000 Apr;182(8):2307-10.
doi: 10.1128/JB.182.8.2307-2310.2000.

A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis

Y Masaoka  1 , Y Ueno, Y Morita, T Kuroda, T Mizushima, T Tsuchiya
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A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis

Y Masaoka et al. J Bacteriol. 2000 Apr.

Abstract

Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B. subtilis. It also caused an elevated energy-dependent efflux of ethidium in E. coli. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.

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Figures

FIG. 1
FIG. 1
Construction of plasmids carrying ebrA and/or ebrB. The plasmid pBET52 carries normal ebrA and ebrB. Plasmid pBET51 carries intact ebrB and defective ebrA in which two nucleotides were removed from the ClaI site. Plasmid pTS93 carries intact ebrA. Both pBET52 and pBET51 are derivatives of pUC19, and pTS93 is a derivative of pACYC184.
FIG. 2
FIG. 2
Accumulation of ethidium in cells. Energy-starved cells of E. coli KAM3/pUC19 and KAM3/pBET52 were loaded with ethidium bromide. Cellular ethidium was monitored continuously by measuring the fluorescence of ethidium at the excitation and emission wavelengths of 500 nm and 580 nm, respectively. After 1 min (arrow), glucose was added to the cell suspension at a final concentration of 20 mM to energize the cells.
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