SUBCELLULAR-LOCALIZATION OF THE MAJOR AUTOLYSIN, ATL AND ITS PROCESSED PROTEINS IN STAPHYLOCOCCUS-AUREUS

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138-kD a protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracell ular peptidoglycan hydrolases, 51-kDa endo-beta-N-acetylglucosaminidas e (51-kDa GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (62-kDa AM) . To investigate cell-associated bacteriolytic enzymes for atl gene pr oducts, proteins were extracted from the cells as follows, The cells w ere exposed to 3 M LiCl followed by 4% SDS, Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS-stable cell-asso ciated bacteriolytic proteins were extracted without disrupting the ce lls, Exposure to 3 M LiCl released major 138-, 115-, 85-, 62- and 51-k Da bacteriolytic proteins, and subsequent 4% SDS extraction released m ajor 138- and 115-kDa bacteriolytic proteins, These bacteriolytic prot eins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 at l::Tn551), Immunoblotting studies suggest that these are all atl gene products: the 138-kDa protein is an ATL with a cleaved signal sequence ; the 115- and 85-kDa proteins are intermediates; and the 51- and 62-k Da proteins are cell-associated 51-kDa GL and 62-kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are l ocated outside the cell membrane. Differences in extractability and im munoelectron microscopic studies suggest that atl gene products are as sociated with cells in two different ways, LiCl extractable and non ex tractable. We suggest that the 138-kDa ATL undergoes processing throug h intermediate proteins (115- and 85-kDa proteins) to mature as the ac tive cell cluster-dispersing enzymes 51-kDa GL and 62-kDa AM on the ce ll surface.