Regulation of the glpFK operon of Escherichia coli K-12 and characterization of it gene products

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Virginia Tech


The glpF gene, which encodes a cytoplasmic membrane protein that facilitates the diffusion of glycerol into the cell, and the glpK gene, which encodes glycerol kinase, map near minute 88 on the linkage map of Escherichia coli K-12. In the present work, the nucleotide sequence of the 843 base Pair glpF gene, 430 base pairs of the glipK gene, and the intervening sequence between the two genes were determined. The control region for the glpFK operon was identified and sequenced. The gipK gene product was purified to near homogeneity by streptomycin sulfate and ammonium sulfate fractionation with subsequent DEAE Sephadex chromatography. N-terminal amino acid analysis identified the startpoint of translation for the gipK gene. The transcription start site was identified 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were -10 and -35 sequences Similar to the consensus sequences for gram bacterial promoter elements. DNase I footprinting was used to identify two binding sites for the CAMP-cAMP receptor protein (CRP) complex upstream from and overlapping the putative -35 sequence. Four binding sites for the glp repressor were located sequentially along the DNA extending from -89 (relative to the start point of transcription) to within the -10 region. Two additional repressor binding sites were identified within the glpK coding region. Interaction of these operator sites with those in the control region was identified. The affinity of the gilp repressor for the control regions of the glpD, glpACB-gipTQ, and glpFK operons was compared by titration studies using a strain harboring a glpT:lacZ fusion and a glpRn mutation.