Charaterization of the glpEGR operon of Escherichia coli K-12

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Virginia Tech


In E. coli, three open reading frames (ORF) are present in the glpEGR operon. The glpR-encoded repressor governs the universal negative regulation of the glp regulon. The glpE and glpG genes are located upstream of glpR. The initiation codons as well as the translational reading frames used by these two genes were determined. Both proteins were successfully overexpressed and GlpE was purified in one step using immobilized metal affinity chromatography. The translational reading frame of glpR in its C-terminal coding region was verified, and confirms the nucleotide sequence reported by other workers. The subcellular locations of GlpE and GlpG were determined, GIpE was in the cytoplasmic fraction and is predicted to be an acidic protein with a molecular weight of 12,082; GlpG was in the cytoplasmic membrane fraction and is predicted to be a basic, hydrophobic protein with a molecular weight of 31,278. glp repressor was also overexpressed and purified. The apparent dissociation constant of the glp repressor-operator complex was estimated to be 50 nM, which indicates that GlpR has a relatively low affinity for a single glp operator site. Isoelectric focusing gel electrophoresis suggested that GlpR exists as three differentially-charged forms in vitro. The major form of GlpR had an isoelectric point of 5.99, and the two minor forms had pl’s of 5.92 and 6.05, respectively. The presence and relative amounts of these forms, however, was not dependent on GlpE/GlpG.

The effect of GlpE/GlpG together with GlpR on the regulation of glp operons was studied using an in vivo system that employed compatible expression vectors for GlpE/GlpG and GlpR. Comparison of the patterns of regulation of glpK, glpD and glpT by the glp repressor agreed well with results previously reported on differential repression of these operons. Under the conditions employed, the glp repressor conferred more than 90-fold repression on glpK. Interestingly, in the presence of GlpE and GlpG, the repression ratio for glpK increased to near 170-fold. In contrast, glpD was subject to 23- fold repression by GlpR. The presence of GlpE and GlpG, however, decreased this ratio to 12-fold. The glpTQ operon was subject to relatively weak repression, about 9- to 12- fold, and repression was independent of the glpE/glpG gene products. This is the first time that the effect of the GlpE/GlpG proteins on the regulation of individual glp operons has been examined. The results suggest that GlpE/GlpG exert effects that bring about differential regulation of glpK, glpD, and glpT. Since the precise biochemical functions of GlpE and GlpG remain undetermined, the molecular basis for this effect is still unknown.