Abstract
Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages phi11, 80alpha, 187, Twort, and phiPVL harbor a central domain that displays sequence homology to known N-acetylmuramyl-L-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the phi11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the phi11 enzyme has D-alanyl-glycyl endopeptidase as well as N-acetylmuramyl-L-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, phi11(Delta181-381), revealed that the D-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.
Highlights
The cell wall envelope of Gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites [1]
In this paper we analyzed the enzymatic properties of 11 murein hydrolase using both staphylococcal peptidoglycan as well as solubilized surface protein as substrates
We conclude that the enzyme is bifunctional, having both an N-acetylmuramyl-L-alanyl amidase and a D-alanyl-glycine endopeptidase activity
Summary
Strains and Materials—Staphylococcal strain OS2 has been previously described [31] and was used as a host for the recombinant plasmid pHTT4 encoding the recombinant surface protein Seb-MH6-Cws [36]. Isolation of Seb-MH6-Cws from Staphylococcal Cell Walls—The purification of Seb-MH6-Cws surface protein from cell walls of S. aureus OS2 harboring the plasmid pHTT4 was carried out as described previously [35]. Digestion of cell walls with mutanolysin was carried out in 100 mM sodium phosphate buffer, pH 6.0, as described previously [35]. S. aureus OS2 cells were grown to mid-log phase in tryptic soy broth, and culture was chilled to 4 °C on ice. Cells were harvested by centrifugation, suspended in a solution of 4% sodium dodecyl sulfate (SDS), and boiled for 30 min. Broken cell walls were collected by centrifugation at 30,000 ϫ g for 15 min, and the pellet was suspended in 50 mM Tris-HCl, 10 mM MgCl2, pH 7.5. Numbers in parentheses indicate the number of fragmentation events necessary to generate the proposed ions
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