IDENTIFICATION OF ENDO-BETA-N-ACETYLGLUCOSAMINIDASE AND N-ACETYLMURAMYL-L-ALANINE AMIDASE AS CLUSTER-DISPERSING ENZYMES IN STAPHYLOCOCCUS-AUREUS

Two proteins which are capable of dispersing sell clusters of Staphylo coccus aureus have been purified from a S. aureus FDA209P culture supe rnatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunob lot) analysis, one Of them was identified as a 51-kDa endo-beta-N-acet ylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidogl ycan fragments following treatment with the 62-kDa protein indicated t hat this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitr o studies of cluster dispersion activities using S. aureus mutant stra ins Lyt66 or S. aureus Wood46 grown as clusters demonstrated that thes e two enzymes act synergistically to disperse clusters into single cel ls. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunogl obulin G induced giant clusters. Clusters induced by anti-51-kDa GL an d by Cibacron blue F3G-A were dispersed by coincubation with the 51-kD a GL and the 62-kDa AM. Western blot analysis demonstrated that the 51 -KDa GL and the 62-kDa AM were missing in culture supernatants of S. a ureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), wh ich grow in clusters. These results strongly suggest that the 51-kDa G L and 62-kDa AM are involved in cell separation of daughter cells afte r cell division.