Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12

Abstract

The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNase I footprinting. The region protected by the cAMP.CRP complex contained tandem sequences resembling the consensus sequence for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream of the start of transcription. The glp repressor protected an extensive area (-89 to -7 relative to the start point of transcription), sufficient for the binding of four repressor tetramers. Two additional binding sites for the repressor were identified within the glpK coding region. The DNA containing these two operators synergistically increased the apparent affinity of glp repressor for DNA fragments containing the four operators in the promoter region of the glpFK operon. With this study, a total of 13 operators for the glp regulon have been characterized. Comparison of these operators revealed the consensus 5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity of the glp repressor for the various glp operators was assessed in vivo using a promoter-probe vector. The relative apparent affinity of the control regions for glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ. The degree of catabolite repression for each of the operons was assessed using a similar system. In this case, the relative sensitivity of the glp operons to catabolite repression was glpTQ greater than glpFK greater than glpACB greater than glpD.