THE 2-COMPONENT LYSIS SYSTEM OF STAPHYLOCOCCUS-AUREUS BACTERIOPHAGE TWORT - A LARGE TTG-START HOLIN AND AN ASSOCIATED AMIDASE ENDOLYSIN

The lysis genes of the virulent Staphylococcus aureus bacteriophage Tw ort were cloned and their nucleotide sequences determined. The endolys in gene plyTW encodes a 53.3-kDa protein, whose catalytic site is loca ted in the amino-terminal domain. An enzymatically active fragment (N- terminal 271 amino acids) was overexpressed in Escherichia coli and pa rtially purified. The enzyme rapidly cleaves staphylococcal peptidogly can, and was shown to act as N-acetylmuramoyl-L-alanine amidase (EC 3. 5.1.28). Significant sequence homology to the specific cell wall targe ting domain of lysostaphin was observed in a 101-amino acid C-terminal overlap. However, we found that the large C-terminal portion (63%, 29 5 aa) of PlyTW is not required for staphylolytic activity. Located ups tream of and overlapping plyTW by 35 bp in a different reading frame ( +1), we identified holTW, which starts with a single TTG tripler. The gene specifies a 185-amino acid (20.5 kDa) holin protein, which featur es two potential hydrophobic, antiparallel transmembrane domains, and a highly charged, acidic C-terminus. HolTW is the largest class II hol in described to date. It can substitute for the defective allele in ph age lambda S amber mutants, both in trans from an expression plasmid, and from within gt11::holTW. The proposed function is the formation of unspecific membrane lesions to promote access of the endolysin to the bacterial peptidoglycan. (C) 1998 Federation of European Microbiologi cal Societies. Published by Elsevier Science B.V.