On the rate limiting step in downhill transport via the LacY permease of Escherichia coli

Abstract

Strains of Escherichia coli K12 were constructed for the specific purpose of evaluating the inducibility of the influx mechanism controlled by the lacY gene. These strains are heteromerodiploids characterized by a high and relatively constant level of beta-D-galactosidase which is not affected significantly by induction of the Lac operon. These properties were obtained by introducing episomal lacI+,Oc,Z+,Y-genes into the cells. In these merodiploids the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) hydrolysis of extracted cells is 50-times that of intact cells. This difference indicates that the rate limiting step in the ONPG hydrolysis by intact cells is influx. Using a set of merodiploids with and without the LacY transport system, we were able to demonstrate a specific induction of ONPG influx. However, the increase in influx due to induction was only 3.5-fold as compared to the 40-fold increase observed when the LacY permease was measured by intracellular accumulation of [14C]TMG.