The nucleotide sequence of the glpEGR operon of Escherichia coli was determined. The translational reading frame at the beginning, middle, and end of each gene was verified. The glpE gene encodes an acidic, cytoplasmic protein of 108 amino acids with a molecular weight of 12,082. The glpG gene encodes a basic, cytoplasmic membrane-associated protein of 276 amino acids with a molecular weight of 31,278. The functions of GlpE and GlpG are unknown. The glpR gene encodes the repressor for the glycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048. The amino acid sequence of the glp repressor was similar to several repressors of carbohydrate catabolic systems, including those of the glucitol (GutR), fucose (FucR), and deoxyribonucleoside (DeoR) systems of E. coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-positive bacteria and agrocinopine (AccR) system of Agrobacterium tumefaciens. These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and bind a sugar phosphate molecule as the inducing signal. The DNA recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive recognition helix of the glp repressor was changed by site-directed mutagenesis to match that of the deo repressor or, in a separate construct, to abolish DNA binding. Neither altered form of the glp repressor recognized the glp or deo operator, either in vivo or in vitro. However, both altered forms of the glp repressor were negatively dominant to the wild-type glp repressor, indicating that the inability to bind DNA with high affinity was due to alteration of the DNA-binding domain, not to an inability to oligomerize or instability of the altered repressors. For the first time, analysis of repressors with altered DNA-binding domains has verified the assignment of the helix-turn-helix motif of the transcriptional regulators in the deoR family.The genes of the glp regulon of Escherichia coli encode the proteins needed for the dissimilation of sn-glycerol 3-phosphate (glycerol-P) and its precursors, glycerol and glycerophosphodiesters (27, 28). The five operons that constitute the glp regulon are located at three different positions on the chromosome. Transcription of the glp operons is subject to multiple controls, including catabolite repression mediated by cyclic AMP-CRP and respiratory control mediated by the FNR and ArcA/ArcB systems (19,28). In addition, each of the operons is negatively controlled by a repressor specific for the regulon, the glp repressor. The extent of repression is different for each operon. Repression is relieved in the presence of the inducer for the regulon, glycerol-P (28).The glpTQ and glpACB operons, located near 51 min of the linkagemap,encodetheglycerol-Ppermease/glycerophosphodiesterase and the subunits of the anaerob...