(A) Representation of the post-translational cleavage (black arrows) of the atl encoded protein. The processed amidase (AM, purple arrow) releases peptidoglycan (PGN) stem peptides by cutting the bond between the stem peptides (black circles) and the N-acetylmuramic acid residue (open square). The glucosaminidase (GL, blue arrow) releases muropeptides by cutting the glycosidic linkage between N-acetylmuramic acid (open square) and N-acetylglucosamine (open circle). For simplicity, only some examples of AM and GL cleavage sites are indicated by arrows. (B) Zymogram analysis of the activity of autolysins extracted from different Staphylococcus aureus atl mutant strains expressing inactive amidase (AMH265A), inactive glucosaminidase (GLE1128A), or both (AMH265A GLE1128A). Top: Cells were harvested at mid-exponential phase, and protein extracts were prepared and run in an SDS–PAGE gel containing Micrococcus luteus cell walls. PGN hydrolytic activity is seen as clear bands in the stained gel and showed that S. aureus encoding mutant proteins AM or GM were lacking only the expected activity. Middle: A similar analysis was carried out with purified proteins AM, GL, and their inactive forms, AMH265A and GLE1128A, respectively, showing that mutant proteins were not active. Bottom: SDS–PAGE of the protein samples loaded into the zymograms confirmed that similar amount of proteins were used. (C) S. aureus parental and atl mutant strains were incubated for mCherry_PGRP-SA in 96-well plates. mCherry_PGRP-SA bound to each bacterial strain is represented as the median with 25% and 75% inter-quartile range (n = 50 wells). A significant increase of mCherry_PGRP-SA binding, relative to the parental strain, was observed with S. aureus mutants expressing AMH265A and AMH265A GLE1128A (p<0.0001), but not the GLE1128A mutant. mCherry_PGRP-SA binding to S. aureus parental and atl mutant strains was also confirmed by fluorescence microscopy. Gray panels are phase-contrast images of bacterial cells (white scale bar represents 1 μm) and black panels show mCherry_PGRP-SA binding.