Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa

Abstract

Two new genes (mexXY) similar to mexAB, mexCD, and mexEF and mediating multidrug resistance were cloned from the chromosome of Pseudomonas aeruginosa. Elevated ethidium extrusion was observed with Escherichia coli cells harboring the plasmid carrying mexXY. This MexXY system confers higher resistance to fluoroquinolones than the MexAB and MexCD systems, and E. coli ToIC or P. aeruginosa OprM is necessary for the function of the MexXY system.

FIG. 1

Accumulation of ethidium in host cells and in transformed cells. E. coli KAM3/pBR322 and KAM3/pTEM4 cells were grown in L medium supplemented with 40 mM potassium lactate. Ethidium bromide was added to cell suspensions of KAM3/pBR322 and KAM3/pTEM4 at a final concentration of 10 μM. Accumulation of ethidium was monitored continuously by measuring the fluorescence of ethidium in cells, at the excitation and emission wavelengths of 500 and 580 nm, respectively. After 7.5 min (arrow), CCCP was added to the suspensions at a final concentration of 100 μM.

FIG. 2

Restriction map of DNA insert carried on pTEM4. The DNA region derived from the P. aeruginosa chromosomal DNA and carried on pTEM4 is shown as a horizontal solid bar. Restriction sites on pTEM4 are indicated. Locations of mexX and mexY are indicated by open arrows.