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 a nd glucose activation in many Gram-positive bacteria, has been purifie d from Bacillus megaterium after fusing it to a His tag. CcpA-his immo bilized on a NI-NTA resin specifically interacted with HPr phosphoryla ted at seryl residue 46. HPr, a phosphocarrier protein of the phosphoe nolpyruvate: glycose phosphotransferase system (PTS), can be phosphory lated at two different sites: (i) at His-15 in a PEP-dependent reactio n catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-depen dent reaction catalysed by a metabolite-activated protein kinase. Neit her unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doub ly phosphorylated HPr bound to CcpA, The interaction with seryl-phosph orylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive b acteria is a protein kinase-triggered mechanism. Glycolytic intermedia tes, stimulating the corresponding protein kinase and the P-ser-HPr/Cc pA complex formation, provide a link between glycolytic activity and c arbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carb on catabolite repression and PTS transport activity.