title: Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals.
abstract: The gene (aur) encoding the metalloprotease (aureolysin) of Staphylococcus aureus from domestic animals was analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The aur gene was detected in all 74 isolates from cows, pigs and chickens by PCR amplification and was classified into types I and II by PCR-RFLP patterns. The type II aur gene was contained in 36 (94.7%) of 38 protease-positive isolates as judged by skim milk agar plate culture, while type I was contained in 28 (77.8%) of 36 protease-negative isolates. The deduced amino acid sequences of aureolysins from type I and II isolates were almost identical with those of the published data. Subsequently, the two aureolysins were purified from the culture supernatants of type I and II isolates. The molecular weights of purified type I and II aureolysins were both estimated at 34kDa by SDS-polyacrylamide gel electrophoresis. These aureolysins had maximum proteolytic activity at 30-50 degrees C and pH 7.0-8.0. Their activity was inhibited by metal- and zinc-specific inhibitors, such as EDTA, EGTA and 1,10-phenanthroline. Specific activity (activity/protein) of type II aureolysin was two times higher than that of type I. These results indicated that the aur gene is highly conserved with two allelic forms (types I and II) among bovine, porcine and avian isolates of S. aureus.
authors.affiliation: Faculty of Biotechnology, Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka, Fukui 910-1195, Japan. takeuchi@fpu.ac.jp
authors.forename: Shotaro
authors.initials: S
authors.lastname: Takeuchi
authors.forename: Minoru
authors.initials: M
authors.lastname: Saito
authors.forename: Keisuke
authors.initials: K
authors.lastname: Imaizumi
authors.forename: Toshio
authors.initials: T
authors.lastname: Kaidoh
authors.forename: Hiroki
authors.initials: H
authors.lastname: Higuchi
authors.forename: Sachiko
authors.initials: S
authors.lastname: Inubushi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000829:Animals, Domestic; D001426:Bacterial Proteins; D001483:Base Sequence; D002417:Cattle; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012150:Polymorphism, Restriction Fragment Length; D013203:Staphylococcal Infections / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D013552:Swine
chemical_list: D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C115504:aureolysin