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 phospho carrier protein of the phosphotransferase system. ATP-dependent phosph orylation of HPr at Ser-16 is required for carbon catabolite repressio n as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosph orylatable alanyl residue are resistant to carbon catabolite repressio n. We here demonstrate that mutation of His-15 of HPr, the site of pho sphoenolpyruvate-dependent phosphorylation, also prevents carbon catab olite repression of the gnt operon. A strain which expressed two mutan t HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in w hich His-15 is replaced by Ala [H15A HPr] on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant H Pr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles se ryl-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 phosphoryl ation sites, had no such effect. In vitro assays substantiated these f indings and demonstrated that in contrast to the wild-type seryl-phosp horylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutan t HPr and H15E, S46D doubly mutated HPr did not interact with CcpA. Th ese results suggest that His-15 of HPr is important for carbon catabol ite repression and that either mutation or phosphorylation at His-15 c an prevent carbon catabolite repression.