The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage ϕWMY: Cloning, sequencing, expression, and mutational analysis in Escherichia coli

• Published in: Gene Vol. 351; pp. 97 - 108
• Main Authors: Yokoi, Ken-ji, Kawahigashi, Nobutaka, Uchida, Maiko, Sugahara, Kazuki, Shinohara, Masayuki, Kawasaki, Ken-Ichi, Nakamura, Shogo, Taketo, Akira, Kodaira, Ken-Ichi
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 23-05-2005
• Subjects: Amidase; Endolysin; Holin; Staphylococcus warneri M; Holin; Endolysin; CBB; RBS; SDS; Staphylococcus warneri M; Amidase; IPTG; PAGE; CHAP; PCR; SH3
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2005.03.006

From the genome library of
Staphylococcus warneri M, the two successive cell-lysis genes (
holWMY and
lytWMY) were cloned and characterized. The
lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and p
I were 54 kDa and 8.95, respectively. When overproduced in
Escherichia coli,
lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative
N-acetylmuramoyl-
l-alanine amidases encoded by the staphylococcal phages ϕ11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for
d-alanyl-glycyl endopeptidase), amidase (
l-muramoyl-
l-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys–His–Asn triad is crucial for the enzymatic function. On the other hand, the
holWMY gene encoded a protein (HolWMY) with molecular mass and p
I of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the
S. aureus phage, ϕ11, 80 alpha, and Twort. Upon mitomycin C exposure of
S. warneri M, a prophage (ϕWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the
holWMY–lysWMY genes in the phage genome.