Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene

Abstract

LacY mutants (535) were isolated from an E. coli strain carrying the lactose operon on an F'' factor, without mutagenesis or after mutagenesis with 2-aminopurine or N-methyl-N''-nitro-N-nitrosoguanidine. Crosses against 48 independently isolated deletions ending in the lacY gene divided the gene into 36 deletion groups. Suppressibility studies with 7 nonsense suppressor strains classified 276 mutants as nonsense mutants and 78 as missense (or nonsuppressible) mutants. Mutants (179) were leaky and could not be so allocated, and 2 had small internal deletions. Nonsense mutants could in many cases be subdivided even within deletion groups on the basis of their suppressibility pattern, giving a total of 70 groups of nonsense mutants. Studies of these mutants allow the following conclusions: lactose and melibiose most probably do not have separate binding sites on the permease; the lacY region most likely consists of 1 cistron, and so both active transport and facilitated diffusion are functions of 1 protein; and there is probably no small defined region of the permease responsible for energy coupling of transport.