Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals.
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.
Takeuchi
Shotaro
S
Faculty of Biotechnology, Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka, Fukui 910-1195, Japan. takeuchi@fpu.ac.jp
Saito
Minoru
M
Imaizumi
Keisuke
K
Kaidoh
Toshio
T
Higuchi
Hiroki
H
Inubushi
Sachiko
S
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
D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C115504:aureolysin