Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K-12

The adjacent, divergently transcribed glpACB and glpTQ operons of Escherichia coli encode the anaerobic glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate transporter/phosphodiesterase, respectively. These operons are negatively controlled by glp repressor binding to operators that overlap the glpA promoter elements. Using DNase I footprinting, three additional operators (O(T)1-3) were identified at positions +307 to +359 within the glpT coding region. To assess a potential regulatory role for these remote operators in vivo, a glpT-lacZ transcriptional fusion containing all of the glpA and glpT operators was constructed. The response of this fusion to the glp repressor was compared to fusion constructs in which O(T)1 and O(T)3 were inactivated, either by deletion or by site-directed mutagenesis. It was found that repression of glpT conferred by binding of glp repressor to glpA operators was increased about three- to fourfold upon introduction of the remote glpT operators. In addition, two integration host factor (IHF) binding sites were identified downstream of the glpT transcriptional start site at positions +15 to +51 and +193 to +227. A regulatory role for IHF was demonstrated by showing that repression of glpT mediated by GlpR was decreased about twofold in strains deficient in IHF and that mutations in IHF1 and/or IHF2 decreased repression about two- to threefold. The effect of IHF was apparent only when the remote operators were present. All of the results are consistent with a model of repression involving GlpR binding simultaneously to the glpA and remote glpT operators, with intervening DNA forming a loop.