Regulation of the activity of the Bacillus subtilis antiterminator LicT bymultiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation

The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon, LicT is inacti ve in mutants affected in one of the two general components of the phosphoe nolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine- containing protein (HPr), We demonstrate that LicT becomes phosphorylated i n the presence of PEP, enzyme I and HPr, The phosphoryl group transfer betw een HPr and LicT is reversible, Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylam ide-urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [P-32]-PEP, enzyme I and HPr, proteolytic digestion of [P-32]-P-LicT, separation of the peptid es by reverse-phase chromatography, mass spectrometry and N-terminal sequen cing of radiolabelled peptides, three histidyl residues were found to be ph osphorylated in LicT, These three histidyl residues (His-159, His-207 and H is-269) are conserved in most members of the BglG/SacY family of transcript ional antiterminators. Phosphorylation of LicT in the presence of serylphos phorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylatio n in the presence of HPr, The slower phosphorylation in the presence of P-S er-HPr leading to reduced LicT activity is presumed to play a role in a rec ently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon.