Protein kinase‐dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram‐positive bacteria

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Abstract
Summary: 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 phosphocarrier 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.

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