Time courses of active lactose transport by E. coli T184
(Z−Y+) or
downhill lactose translocation by E. coli HB101
(Z+Y−)
expressing wild-type permease, mutants R302A and R302S, no permease
(pT7–5 vector only), or mutant E325A. Cells were grown at 37°C, and
aliquots of cell suspensions (50 μl, containing ≈35 μg of protein
in 100 mM KPi, pH 7.5/10 mM MgSO4) were
assayed at 0.4 mM final external lactose concentrations as described in
Experimental Procedures. For downhill assays with
E. coli HB101, 20 μM CCCP was included to abolish the
H+ electrochemical gradient.