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

Abstract

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.

Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus

“…The erythromycin (Em) resistance gene ermB was isolated from plasmid pI258 [14] and subsequently introduced in the plasmids pUC18, pUC19 or pBluescript KS + (Stratagene). Plasmid pUC19‐ ptsI served as the source of the phosphotransferase gene ptsI from S. carnosus [15]. The sucrose utilization plasmid pRA7 [16] was used to inactivate the scrB gene of S. xylosus .…”

Structure of the Full-length Enzyme I of the Phosphoenolpyruvate-dependent Sugar Phosphotransferase System

“…Protein Expression and Purification-EI from S. carnosus was overexpressed in Escherichia coli (strain DH5␣) harboring the plasmid pUC-ptsO2.6X (23). Cells were grown in the presence of isopropyl-␤-D-thiogalactoside in TBY broth (10 g of Tryptone, 5 g of NaCl, 5 g of yeast extract/liter).…”

“…6 The atomic coordinates have been deposited with the Protein Data Bank (www.rcsb.org), under accession code 2HRO. (23) was overexpressed and purified as described under "Experimental Procedures." Initial hexagonal crystals appearing in Hampton Standard screens diffracted poorly, whereas monoclinic crystals, appearing 3-5 days later, diffracted up to 2.5 Å.…”

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon

“…EI of the PTS was purified from Staphylococcus carnosus and HPr was purified from B. subtilis as described before (16,24). For the purification of LevR, the levR gene was cloned under the control of the strong degQ36 promoter (39).…”