Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.

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.