title: Bacteriophage 933W encodes a functional esterase downstream of the Shiga toxin 2a operon.
abstract: In this study, the 1938bp open reading frame z1466, which is encoded directly downstream the Shiga toxin 2a (Stx2a) operon in E. coli O157:H7 phage 933W was cloned and expressed recombinantly. Purification with Ni-NTA agarose beads with subsequent SDS-PAGE revealed a 68kDa protein, designated 933Wp42-His. Analysis of 933Wp42-His demonstrated an esterase activity by activity staining of native gels using triacetin as a substrate. Purified 933Wp42-His demonstrated a Km value of about 10mM and a Vmax value of 1.667nkat/ml for 4-methylumbelliferyl-acetate (4-MUF-Ac) as a substrate. The enzyme was most active in the pH-range of 7.0-8.0, and at 50°C. Furthermore, 933Wp42-His was able to hydrolyze acetic acid from mucin, and 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). This is the first description of an enzymatic activity of the Stx-phage-encoded protein 933Wp42. Its role in substrate utilization during colonization and human infection is discussed.
authors.affiliation: Department of Food Microbiology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 28, 70599 Stuttgart, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Nübling
authors.affiliation: Department of Biotechnology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 25, 70599 Stuttgart, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eisele
authors.affiliation: Department of Food Microbiology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 28, 70599 Stuttgart, Germany.
authors.forename: Helen
authors.initials: H
authors.lastname: Stöber
authors.affiliation: Department of Food Microbiology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 28, 70599 Stuttgart, Germany.
authors.forename: Joschua
authors.initials: J
authors.lastname: Funk
authors.affiliation: Department of Food Microbiology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 28, 70599 Stuttgart, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Polzin
authors.affiliation: Department of Biotechnology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 25, 70599 Stuttgart, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
authors.affiliation: Department of Food Microbiology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 28, 70599 Stuttgart, Germany. Electronic address: herbert.schmidt@uni-hohenheim.de.
authors.forename: Herbert
authors.initials: H
authors.lastname: Schmidt
mesh_terms: D002846:Chromatography, Affinity; D003090:Coliphages / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019453:Escherichia coli O157 / Q000821:virology; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009876:Operon; D022621:Shiga Toxin / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature; D014468:Umbelliferones / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D014468:Umbelliferones; D014764:Viral Proteins; C032078:4-methylumbelliferyl acetate; D022621:Shiga Toxin; D004950:Esterases
keywords: 5-N-acetyl-9-O-acetyl neuraminic acid; 933Wp42; Bacteriophage 933W; Enzyme activity; Escherichia coli O157:H7; Esterase; Mucin; Shiga toxin; yjhs; z1466