CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli.
The cellular cAMP level is markedly down-regulated by cAMP receptor protein (CRP) in Escherichia coli. CRP regulates adenylate cyclase both at the level of transcription of its structural gene cya and at the level of enzyme activity. We established a method to determine the phosphorylation state of IIA(Glc), the glucose-specific phosphotransferase protein, in intact cells. We found that IIA(Glc) exists predominantly in the unphosphorylated form in wild-type cells growing in LB medium, while it is largely phosphorylated in crp or cya cells. Disruption of the ptsG gene that codes for the membrane component of the major glucose transporter (IICB(Glc)), and/or the fruF gene coding for FPr (fructose-specific hybrid phosphotransferase protein), did not affect the phosphorylation state of IIA(Glc). When IICB(Glc) was overproduced in the presence of glucose, the levels of both cAMP and phosphorylated IIA(Glc) in crp cells were concomitantly decreased to wild-type levels. In addition, when His-90 in IIA(Glc) was replaced by glutamine, both phosphorylation of IIA(Glc) and the overproduction of cAMP in crp cells were eliminated. We also found that extracts of crp+ cells markedly stimulate dephosphorylation of IIA(Glc)-P in vitro. We conclude that CRP-cAMP down-regulates adenylate cyclase primarily by reducing the level of phosphorylated IIA(Glc). The data suggest that unspecified proteins whose expression is under the control of CRP-cAMP are responsible for this regulation.
Takahashi
H
H
Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa, Japan.
Inada
T
T
Postma
P
P
Aiba
H
H
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017384:Sequence Deletion
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; C032242:crr protein, E coli; D006639:Histidine; D000242:Cyclic AMP; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D000262:Adenylyl Cyclases; D005947:Glucose