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 phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified 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 relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 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 Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation 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 phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.
Parche
S
S
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Schmid
R
R
Titgemeyer
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000378:metabolism; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000235:genetics; D013302:Streptomyces / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; C030182:phosphocarrier protein HPr; C031629:phosphoenolpyruvate-glucose phosphotransferase; C029022:phosphoenolpyruvate-lactose dependent phosphotransferase system; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase