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 pts H 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 Staph ylococcus carnosus. The restriction fragment was cloned in the antisen se orientation to the lac promoter in the low-copy-number vector pSU18 . The nucleotide sequences of the ptsI gene, which encodes enzyme I (E C 2.7.3.9), and the corresponding flanking regions were determined. Th e primary translation product, derived from the nucleotide sequence, c onsists 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 histid inyl residue at position 191 could be identified as the probable phosp hoenolpyruvate-dependent phosphorylation site of enzyme I of S. carnos us because of sequence homologies with the peptide sequences of enzyme I-active sites of Enterococcus faecalis and Lactococcus lactis. Sever al 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 ou t. The generation times and interaction between enzyme I with histidin e-containing protein from gram-positive and gram-negative bacteria wer e measured in a phosphoryl group transfer test.