Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsI gene and expression and complementation studies of the gene product.
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.
Kohlbrecher
D
D
Department of Microbiology, Ruhr-Universität Bochum, Germany.
Eisermann
R
R
Hengstenberg
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D016415:Sequence Alignment; D013210:Staphylococcus / Q000235:genetics*
D004269:DNA, Bacterial; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System