Solute-binding activity and competitive inhibition tests using the fluorescent substrates EtBr (10 μM) and SYBR green (100 U) and various concentrations of ampicillin. The excitation (Ex) and emission (Em) wavelengths were 544 and 590 nm for EtBr and 485 and 538 nm for SYBR green, respectively. (A) Transport of EtBr in wild-type V. furnissii (•) or VFYKW1 (○). (B) Transport of EtBr in YKWEC1 or YKWEC2. □, EtBr alone; ▵, pET28a(+) alone; ⋄, pYKW1 alone; ○, pET28a(+) plus EtBr; •, pYKW1 plus EtBr. (C) Transport of SYBR green in wild-type V. furnissii (•) or VFYKW1 (○). (D) Transport of SYBR green in YKWEC1 or YKWEC2. ○, pET28a(+) plus SYBR green; •, pYKW1 plus SYBR green. (E) Ampicillin competitively inhibited transport of EtBr in YKWEC1 in a concentration-dependent manner. ▴, pYKW1 plus EtBr; •, pYKW1 plus EtBr plus 0.5 mg/ml ampicillin; ○, pYKW1 plus EtBr plus 2 mg/ml ampicillin. (F) SYBR green competitively inhibited transport of EtBr in YKWEC1 in a concentration-dependent manner. ▴, pYKW1 plus EtBr; •, pYKW1 plus EtBr plus 5 U SYBR green; ○, pYKW1 plus EtBr plus 50 U SYBR green.