Deconstructing glucose-mediated catabolite repression of the lac operon of Escherichia coli: I. Inducer exclusion, by itself, cannot account for the repression.

Abstract

The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to CRP-mediated transcriptional inhibition and EIIAGlc-mediated inducer exclusion. The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. The literature shows that in fully induced cells, inducer exclusion reduces the permease activity only 2-fold. However, it is conceivable that inducer exclusion drastically reduces the permease activity in partially induced cells. We measured the decline of lactose permease activity due to inducer exclusion in partially induced cells, but found that the permease activity decreased no more than 6-fold. We show that the repression is small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONS AND NOTATIONScAMP3’,5’-cyclic adenosine monophosphateCGSCColi Genetic Stock Centre, Yale UniversityCRPcAMP receptor proteinEIIAGlcEnzyme III of glucose transport system, PTS (Previously EIIIGlc)gdwGrams of cell dry weight, gIPTGIsopropyl β-D-thiogalactopyranoside or MeSHLBLuria-Bertani brothMUMiller unitsOD600Optical density at 600 nmONPGortho-nitrophenol-β-D-galactopyranosideRNAPRibonucleic acid (RNA) polymeraseTMGMethyl- β-D-1-thiogalactopyranoside[14C]TMGCarbon-14 labelled isotope of TMGUVUltraviolet (radiation)X-gal5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideNOTATIONSIntracellular concentration of phosphorylated EIIAglc in the absence of glucoseIntracellular concentration of dephosphorylated EIIAglc in the presence of glucoseEgSpecific activity of β-galactosidaseSteady state specific activity of β-galactosidase in the presence of glucoseSteady state specific activity of β-galactosidase in the presence of glycerolEpSpecific activity of Lac permeaseSteady state specific activity of Lac permeaseEp,tSpecific activity of free Lac permease in the absence of glucoseφpFractional reduction of permease activity in the presence of glucose, 1 - Ep/Ep,tk1, k2Proportionality constantsKdDissociation constant for complex OD600Optical density at 600 nmSp. rate of β-galactosidase synthesis in the presence of glucose + glycerol + TMGSp. rate of β-galactosidase synthesis in the presence of glycerol + TMGSpecific rate of diffusive efflux of TMGTIntracellular TMG concentrationSteady-state intracellular TMG concentrationSteady-state intracellular TMG concentration in the presence of glucoseSteady-state intracellular TMG concentration in the presence of glucoseTeExtracellular TMG concentrationµglcSpecific growth rate of E. coli cells on glucoseµglySpecific growth rate of E. coli cells on glycerolXBiomass or cell density, gdw l-1View Full Text