Molecular cloning of the crr gene and evidence that it is the structural gene for IIIGlc, a phosphocarrier protein of the bacterial phosphotransferase system.

Abstract

Sugar substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) normally prevent bacterial cells from utilizing sugars that are not substrates of this system (diauxic growth, the glucose effect). This type of PTS-mediated repression can be completely reversed by a single mutation, designated crr. Two lines of evidence are presented in this report showing that crr is the structural gene for IIIGlc, one of the proteins of the PTS. Homogeneous IIIGlc was isolated from wild-type and a crr- mutant of Salmonella typhimurium, and the proteins were compared. The preparations of IIIGlc were indistinguishable, except that IIIGlc from the mutant showed only 2-3% of the activity of the wild-type IIIGlc in its ability to act as a phosphocarrier protein in the in vitro phosphorylation of methyl .alpha.-glucoside. Also, under contain conditions, the 2 proteins exhibited different behavior on gel filtration columns and in polyacrylamide gel electrophoresis. The Escherichia coli crr gene, which has an estimated minimum length of 0.6 kilobase, was cloned into a high-copy-number plasmid as part of a 1.3 kilobase fragment. The plasmid transforms E. coli crr- to crr+ strains and simultaneously directs the synthesis of IIIGlc.