The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli.
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 pI 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 phi11, 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 pI 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, phi11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (phiWMY) 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.
Yokoi
Ken-Ji
KJ
Molecular Biology Group, Faculty of Engineering, Toyama University, Japan.
Kawahigashi
Nobutaka
N
Uchida
Maiko
M
Sugahara
Kazuki
K
Shinohara
Masayuki
M
Kawasaki
Ken-Ichi
K
Nakamura
Shogo
S
Taketo
Akira
A
Kodaira
Ken-Ichi
K
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001433:Bacteriolysis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D023061:Gene Order; D008565:Membrane Proteins / Q000235:genetics*; D008854:Microscopy, Electron; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D017354:Point Mutation; D039002:Prophages / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000187:drug effects / Q000821:virology*; D013204:Staphylococcus Phages / Q000235:genetics* / Q000648:ultrastructure; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000648:ultrastructure
D004279:DNA, Viral; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D016685:Mitomycin; D010450:Endopeptidases; C001143:endolysin; D000581:Amidohydrolases