The phosphotransferase system (PTS) of Streptomyces coelicolor - Identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH

HPr, the histidine-containing phosphocarrier protein of the bacterial phosp hotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purifi ed HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relations hip revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 1 5 is perfectly conserved while a second possible phosphorylation site at se rine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Stap hylococcus aureus, respectively. This phosphoenolpyruvate-dependent phospho rylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phospho rylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIA(Mannitol) of E. coli. ATP-dependent phosphorylation was detected with H Pr kinase/phosphatase of B. subtilis. These results present the first ident ification of a gene of the PTS complement of S. coelicolor, providing the b asis to elucidate the role(s) of HPr and the PTS in this class of bacteria.