Background : The inhibition of¯-galactosidase expression in glucose-lactose diauxie is a typical example of the glucose effect in Escherichia coli. It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level. However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose-lactose system.

SummaryGlucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at ¹40.5 and ¹95.5 and that Mlc binds at two regions centred around ¹8 and ¹175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRPcAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.

We report a novel post-transcriptional control of the ptsG gene encoding the major glucose transporter IICB Glc . We demonstrate that the level of IICB Glc is markedly reduced when the glycolytic pathway is blocked by a mutation in either the pgi or pfkA gene encoding phosphoglucose isomerase or phosphofructokinase, respectively. This down-regulation of ptsG is not exerted at the transcriptional level. Both northern blot and S1 analyses demonstrate that the mutation dramatically accelerates the degradation of ptsG mRNA. The degradation of ptsG mRNA occurs in wild-type cells when a-methylglucoside, a nonmetabolizable analog of glucose, is present in the medium. The addition of any one of the glycolytic intermediates downstream of the block prevents the degradation of ptsG mRNA. The rapid degradation of ptsG mRNA is eliminated when RNase E is thermally inactivated. We conclude that the glycolytic pathway controls ptsG expression by modulating RNase Emediated mRNA degradation. This is the ®rst instance in which the glycolytic¯ux has been shown to affect the expression of a speci®c gene through mRNA stability.

External glucose stimulates transcription of several genes including ptsG encoding IICB(Glc), a membrane component of the phosphotransferase system (PTS), by relieving the negative regulation of a global repressor Mlc in Escherichia coli. We investigate here how glucose modulates Mlc action. The Mlc-mediated repression is eliminated by a ptsI mutation, while Mlc is constitutively active in a ptsG mutant. We show that IICB(Glc)-FLAG interacts physically with Mlc in crude extracts prepared from cells in which IICB(Glc) is supposed to exist as the non-phosphorylated form. The IICB(Glc)-Mlc interaction is no longer observed when IICB(Glc) is phosphorylated. Exogenously added purified Mlc binds to purified IICB(Glc)-FLAG. We also demonstrate that Mlc is associated with membrane when IICB(Glc) is dephosphorylated while it is in the cytoplasm when IICB(Glc) is phosphorylated or absent. We conclude that IICB(Glc) regulates the cellular localization of Mlc, depending on its phosphorylation state, which is determined by the availability of external glucose. Thus, glucose induces the transcription of Mlc-regulated promoters by sequestering Mlc to the membrane through dephosphorylation of IICB(Glc).

The inhibition of ␤-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in Escherichia coli. We studied the glucose effect in the lacL8UV5 promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of ␤-galactosidase expression by glucose and a diauxic growth were observed when the lacL8UV5 cells were grown on a glucose-lactose medium. The addition of isopropyl ␤-D-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the crr gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose-lactose medium is not due to the reduction of CRP-cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the lacL8UV5 cells was no longer observed when either the crp or the cya gene was disrupted. Evidence suggested that CRP-cAMP may not enhance directly the lac repressor action in vivo. Northern blot analysis revealed that the mRNA for ptsG, a major glucose transporter gene, was markedly reduced in a ⌬crp or ⌬cya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in ⌬cya or ⌬crp cells. We conclude that CRP-cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose-lactose diauxie, by activating the expression of the ptsG gene.In enteric bacteria, the synthesis of many catabolic enzymes is inhibited by the presence of glucose in the growth medium. Multiple mechanisms are involved in this phenomenon, referred to as ''glucose effect '' or ''glucose repression'' (1-5). Although glucose signaling may occur via different pathways, glucose ultimately would affect the transcription of catabolic operons by modulating transcription factor(s). In the lactose operon of Escherichia coli, the final targets of glucose are the lac repressor and the positive regulator, the complex of cAMP receptor protein (CRP) and cAMP. First, glucose prevents the entry of inducer into the cell, resulting in an increase in the concentration of the inducer-free lac repressor. The mechanism of this process, called ''inducer exclusion,'' is relatively well understood (3-5). The transport of glucose into the cell by the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) decreases the level of phosphorylation of enzyme IIA Glc , one of the enzymes involved in glucose transport. The dephosphorylated enzyme IIA Glc binds to and inactivates the lac permease, causing the inducer exclusion. Second, glucose lowers the level of CRP-cAMP by reducing the intracellular concentrations of both cAMP and CRP under certain conditions, for example, when added to cells growing on a poor carbon source such as glycerol or succinate (6, 7). Glucose is thought to reduce cAMP level by decreasing the phosphorylated form of enzyme IIA Glc , which is proposed to be invo...