cAMP receptor protein-cAMP plays a crucial role in glucose-lactose diauxie by activating the major glucose transporter gene in Escherichia coli

Abstract

The inhibition of beta-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 beta-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 beta-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 delta crp or delta cya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in delta cya or delta 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.

Figure 1

Growth curve and β-galactosidase activity of lacL8UV5 cells growing on a glucose–lactose medium. Cells were grown in M9 medium containing 0.04% glucose and 0.2% lactose. The following strains and addition were used: (A) PR166; (B) PR166 plus 0.5 mM isopropyl β-d-thiogalactoside; (C) KK21; and (D) PR166 harboring pIT539. At the indicated time, samples were removed to determine the OD (squares) and β-galactosidase activity (diamonds).

Figure 2

Glucose effect in lacL8UV5 Δcrp cells. KK17 (A) or KK17 harboring pHA7 (B) were grown in M9 medium containing 0.04% glucose and 0.2% lactose. At the indicated time, samples were removed to determine the OD (squares) and β-galactosidase activity (diamonds).

Figure 5

Glucose effect and the expression of ptsG gene in lacL8UV5 cells. (A) Northern blot analysis of the ptsG mRNA. Cells were grown in M9 medium containing 0.5% glucose and 0.5% lactose. Total RNAs, 50 μg (lanes 1, 2, 3, 5, 6 and 8) or 5 μg (lanes 4, 7 and 9), isolated from PR166 (lane 1), KK17 (lane 2), KK17 harboring pHA7 (lane 3), KK17 harboring pTH111 (lane 4), KK8 (lane 5), KK8 harboring pIT302 (lane 6), KK8 harboring pTH111 (lane 7), KK20 (lane 8), and KK20 harboring pTH111(lane 8) were subjected to Northern blot analysis. (B) Cells were grown in M9 medium containing 0.5% glucose and 0.5% lactose. β-Galactosidase activity was determined at OD₆₀₀ of 0.6. Each value is the average of three experiments. (C) KK17 cells harboring pTH111 were grown in M9 medium containing 0.04% glucose and 0.2% lactose. At the indicated time, samples were removed to determine the OD (squares) and β-galactosidase activity (diamonds).

Figure 3

DNA probes for Northern blotting experiment. (A) The restriction map of the DNA region around the ptsH, ptsI, and crr genes. (B) The restriction map of the DNA region around the ptsG gene. The data were taken from refs. and , respectively. The open boxes indicate the coding region. Black lines beneath the map represent the DNA probes used for Northern blotting.

Figure 4

Glucose effect and the expression of ptsHI genes in lacL8UV5 cells. (A) Northern blot analysis of the ptsHI mRNA. Cells were grown in M9 medium containing 0.5% glucose and 0.5% lactose. Total RNAs, 50 μg (lanes 1, 2, and 4) or 5 μg (lanes 3 and 5), isolated from PR166 (lane 1), KK17 (lane 2), KK17 harboring pST51 (lane 3), KK8 (lane 4), and KK8 harboring pST51 (lane 5) were subjected to Northern blot analysis. (B) Cells were grown in M9 medium containing 0.5% glucose and 0.5% lactose. β-Galactosidase activity was determined at OD₆₀₀ = 0.6. Each value is the average of three experiments.

Figure 6

A model explaining the role of CRP–cAMP in the glucose effect in the glucose–lactose system. The transport and phosphorylation of glucose by glucose PTS (IIA^Glc + IICB^Glc) increase the dephosphorylated IIA^Glc that prevents the uptake of lactose by inhibiting the lac permease activity. An important role of CRP–cAMP in the glucose effect is to support inducer exclusion by activating the ptsG transcription. The reduction in ptsG transcription is responsible for the failure of the glucose effect in the absence of CRP–cAMP.