Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus

Abstract

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. 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 immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa 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. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.