CHEMOSTAT SELECTION OF AN ESCHERICHIA-COLI MUTANT CONTAINING PERMEASEWITH ENHANCED LACTOSE AFFINITY

Chemostats supplied with limited lactose were used to ask whether it w as possible to generate and isolate any mutant of Escherichia coli lac tose permease which allowed cells to grow faster. The permease and bet a-galactosidase activities of the chemostat culture initially rose tog ether to reach a plateau. After 30 days, the former underwent a second increase alone. From this culture, a faster-growing mutant was isolat ed. Its permease gene was cloned, sequenced, and found to have a singl e base pair changed. Thymine at position 199 was changed to guanine, r esulting in serine 67 being substituted by alanine. Cells bearing this mutant in the plasmid could grow faster than parents in 10 mu M lacto se. The K-m of the mutant permease toward lactose was 1.4 mM, about ha lf of the wild-type value. Thus, a mutant with higher affinity for sub strate could be selected from the chemostat. (C) 1996 Academic Press, Inc.