Design and probing of efflux functions of EGFP fused ABC membrane transporters in live cells using fluorescence spectroscopy.

We have designed and constructed fusion genes of C-terminal (Ct) or N-terminal (Nt) bmrA with EGFP vectors and successfully expressed them in ΔBmrA (BmrA deletion strain of Bacillus subtilis), generating two new strains of B. subtilis (Ct-BmrA-EGFP and Nt-BmrA-EGFP). The fusion genes were characterized using gel electrophoresis and DNA sequencing. Their expression in live cells was determined by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy and spectroscopy. The efflux function of the new strains was studied by measuring their accumulation kinetics of intracellular Hoechst dye molecules (a pump substrate) using fluorescence spectroscopy, which were compared with wild-type (WT-BmrA) and ΔBmrA strains. Both new strains show lower accumulation rates than ΔBmrA, and their efflux kinetics are inhibited by a pump inhibitor (orthovanadate). The results suggest that both strains extrude the dye molecules and the fusion proteins retain the efflux function of BmrA (ATP-binding cassette, ABC, transporter). Notably, Nt-BmrA-EGFP strain shows lower accumulation rates (higher efflux rates) than Ct-BmrA-EGFP. Modeled structures of the fusion proteins illustrate a highly flexible linker region connecting EGFP with BmrA, suggesting a minimal obstruction of EGFP to the BmrA. A closer distance of two C termini (~14 Å) than two N termini (47.9 Å) of the "closed" BmrA dimer depicts the larger steric effect of C-terminal fusion. This study also shows that glucose affects the fluorescence study of efflux function of BmrA, suggesting that efflux kinetics of ABC membrane transporters in live cells must be characterized in the absence of glucose.