Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli

Abstract

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.

FIG. 1.

NorM and YdhE reduce the levels of accumulation of norfloxacin and ethidium bromide in E. coli cells. (a) Accumulation of norfloxacin in cells transformed by NorM (AG100AX/pBADΩnorM), YdhE (AG100AX/pBADΩydhE), or an empty vector (AG100AX/pBAD). CCCP was added to the suspensions (first arrow) at a final concentration of 100 μM. After 15 min, glucose was added (second arrow) at a final concentration of 0.4%. (b) Accumulation of ethidium bromide in the same strains. CCCP was added to the suspensions (first arrow) at a final concentration of 100 μM. After 15 min, glucose was added (second arrow) at a final concentration of 0.4%. *, the values for AG100AX/pBADΩnorM and AG100AX/pBADΩydhE cells were significantly different from those for the control (AG100AX/pBAD) (P < 0.05).

FIG. 2.

Sodium ion reduces the level of accumulation of ethidium bromide in cells transformed by NorM. (a) Accumulation of ethidium bromide in AG100AX/pBADΩnorM cells after the addition of 100 mM NaCl or KCl; (b) accumulation of ethidium bromide in AG100AX/pBAD cells (cells carrying the empty vector) after the addition of 100 mM NaCl or KCl. *, the values of the ethidium bromide fluorescence intensity in the presence of 100 mM NaCl were significantly different from those in the presence of 100 mM KCl (P < 0.01).

FIG. 3.

Sodium ion enhances norfloxacin efflux via NorM. (a) Norfloxacin efflux from AG100AX/pBADΩnorM cells carrying the norM gene from N. gonorrhoeae; (b) norfloxacin efflux from AG100AX/pBAD cells carrying the empty vector. NaCl or KCl was added to the suspensions (arrow) at a final concentration of 100 mM. *, the values of norfloxacin fluorescence in the presence of 100 mM NaCl were significantly different from those in the presence of 100 mM KCl (P < 0.05).

FIG. 4.

Effect of Na⁺ concentration on norfloxacin extrusion via NorM. Norfloxacin efflux from AG100AX/pBADΩnorM cells carrying the norM gene was measured in the presence of 0 to 200 mM NaCl. *, the values of norfloxacin fluorescence in the presence of 50, 100, or 200 mM NaCl were significantly different from those in the absence of NaCl (P < 0.003), and the values of norfloxacin fluorescence in the presence of 100 or 200 mM NaCl were significantly different from those in the presence of 50 mM NaCl (P < 0.01).

FIG. 5.

Representative fluorescence polarization of NorM in 0.02% DDM with rhodamine 6G. (a) Binding isotherm of NorM with rhodamine 6G showing a K_D of 3.4 ± 0.2 μM in buffer containing 20 mM HEPES-NaOH (pH 7.5) and 0.02% DDM. (b) Hill plot of the data obtained for rhodamine 6G binding to NorM. α, the fraction of bound rhodamine 6G. The plot gives a slope of 1.01 ± 0.03, indicating a simple binding process with no cooperativity. The interception of the plot provides a K_D of 3.6 ± 0.1 μM for rhodamine 6G binding.

FIG. 6.

Representative fluorescence polarization of YdhE in 0.02% DDM with rhodamine 6G. (a) Binding isotherm of YdhE with rhodamine 6G showing a K_D of 3.0 ± 0.2 μM in buffer containing 20 mM HEPES-NaOH (pH 7.5) and 0.02% DDM. (b) Hill plot of the data obtained for rhodamine 6G binding to YdhE. α, the fraction of bound rhodamine 6G. The plot gives a slope of 1.00 ± 0.04, indicating a simple binding process with no cooperativity. The interception of the plot provides a K_D of 3.6 ± 0.2 μM for rhodamine 6G binding.

FIG. 7.

Effect of pH on the K_D of rhodamine 6G binding to NorM. The resulting K_Ds were plotted against the pH.

FIG. 8.

Effect of Na⁺ concentration on the K_D of rhodamine 6G binding to NorM. The resulting K_Ds were plotted against the NaCl concentration.

FIG. 9.

YdhE binding competition experiment between rhodamine 6G and TPP. YdhE (5 μM) was preincubated with rhodamine 6G (1 μM) for 2 h before titration. The change in the fluorescence polarization signals (ΔFP) of rhodamine 6G was measured at an emission wavelength of 550 nm. TPP was nonfluorescent under the experimental conditions used. The decrease in the change in the fluorescence polarization signals showed that the bound rhodamine 6G was knocked off by TPP.