Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria.
CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phospho-carrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.
Deutscher
J
J
Institut de Biologie et Chimie des Protéines, CNRS, Lyon, France.
Küster
E
E
Bergstedt
U
U
Charrier
V
V
Hillen
W
W
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002244:Carbon / Q000378:metabolism*; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006019:Glycolysis; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005635:Fructosediphosphates; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D012694:Serine; D006639:Histidine; D002244:Carbon; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C029063:fructose-1,6-diphosphate