The staphylococcal peptidoglycan (PGN) is a major and essential component of the cell wall. It is a dynamic structure that undergoes constant cycles of polymerization and hydrolysis which is carried out by transglycosylases/peptidases and PGN hydrolases. The major autolysin, Atl, is the most predominant PGN hydrolase in staphylococci. Though its structure and function have been studied extensively, little is known about the activity and interplay of its domains AmiA and GlcA during cell division and separation. With this study, a highly resolved complex structure of the catalytic amidase domain of S. aureus Atl was elucidated and its molecular ligand interaction mechanism and natural substrate specificity was resolved. Investigating the deletion mutants of both AmiA and GlcA, as well as AtlA revealed that each of the enzymes plays a significant role in cell aggregation, proper septum formation and cell separation. Notably, loss of GlcA activity results in aberrant septum formation manifested by deformed and in part “kidney” like cell clusters. Despite a lowered peptidoglycan crosslinking, the atl, amiA und glcA mutants were 10,000 to 100,000 times more tolerant to oxacillin than the parent strain. For the first time, this work shows the activity of both AmiA and GlcA on their natural substrate, PGN. We found out that AmiA activity is specific to its host PGN while the naked glycan backbone is the natural substrate of GlcA. Our results revealed that the resolution of PGN during cell separation occurs in a defined order, in which AmiA first hydrolyzes the crosspeptides, followed by GlcA chewing back the naked glycan and releasing disaccharides. Additionally, we disproved the hitherto conclusion and showed that GlcA is not an endo- but an exo-beta-N-acetylglucosaminidase. Advisors/Committee Members: Götz, Friedrich (Prof. Dr.) (advisor).