A digoxigenin-labeled DNA probe that was complementary to the gene ptsH and the beginning of the gene ptsI was used to clone a 3.2-kb HincII-BamHI restriction fragment containing the complete ptsI gene of Staphylococcus carnosus. The restriction fragment was cloned in the antisense orientation to the lac promoter in the low-copy-number vector pSU18. The nucleotide sequences of the ptsI gene, which encodes enzyme I (EC 2.7.3.9), and the corresponding flanking regions were determined. The primary translation product, derived from the nucleotide sequence, consists of 574 amino acids and has a calculated molecular weight of 63,369. Amino acid sequence comparison showed 47% similarity to enzyme I of Escherichia coli and 37% similarity to the enzyme I domain of the multiphosphoryl transfer protein of Rhodobacter capsulatus. The histidinyl residue at position 191 could be identified as the probable phosphoenolpyruvate-dependent phosphorylation site of enzyme I of S. carnosus because of sequence homologies with the peptide sequences of enzyme I-active sites of Enterococcus faecalis and Lactococcus lactis. Several in vivo and in vitro complementation studies with the enzyme I ptsI genes of S. carnosus and the E. coli ptsI mutant JLT2 were carried out. The generation times and interaction between enzyme I with histidine-containing protein from gram-positive and gram-negative bacteria were measured in a phosphoryl group transfer test.

No abstract

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. In Gram-positive Staphylococci and Streptococci the phosphoenolpyruvate-dependent bacterial phosphotransferase system (PTS) represents the main sugar-transport system, catalyzing the vectorial phosphorylation of sugar during transport into the cytoplasm. In these bacteria most sugars are transported by this multienzyme complex, consisting of the two general, constitutively expressed cytoplasmic proteins enzyme I and histidine-containing protein (HPr) and different sugar-specific, inducible and membrane-bound enzyme II/enzyme 111 complexes. In an initial step phosphoenolpyruvate phosphorylates the protein kinase enzyme I in a Mg2+-dependent reaction at the N' atom of a histidine residue. From the phosphorylated enzyme I the phospho group is transferred to the catalytic site of the central phosphocarrier HPr to the N" atom of Hisl5. HPr phosphorylated at His15 serves as a phospho group distributor by phosphorylating the different sugar-specific enzyme III/enzyme I1 complexes. Finally, the phosphorylated enzyme I1 catalyzes the membrane translocation of a sugar molecule under concomitant phosphorylation. For a detailed overview, the reader is referred to one of the recent reviews [l].In contrast to Gram-negative bacteria, it was found that the sugar uptake in Gram-positive bacteria via PTS is regulated by an ATP-dependent HPr kinase [2], phosphorylating Correspondence to W. Hengstenberg,

No abstract

No abstract