FIG 4.

EssH exhibits amidase and endopeptidase activities. (A) Overnight cultures of S. aureus USA300 LAC* were treated with lysostaphin or _STREPEssH, and lysis was measured as a decline in A₆₀₀ over time. (B) Purified S. aureus peptidoglycans were digested with mutanolysin and split into two samples that were either left untreated (black trace) or were treated with purified _STREPEssH (red trace); the carbohydrates in both samples were reduced and analyzed by reversed-phase HPLC. Peaks labeled were desalted and analyzed by MALDI-TOF mass spectrometry; observed m/z are reported in Table 1 alongside their structural interpretation. The asterisk identifies the cross-linked muropeptide dimer of S. aureus (m/z 2,438.59). (C) A sample of purified muropeptide dimer (*) was split into two parts, either left untreated (mock) or treated with _STREPEssH (red trace), and analyzed by reversed-phase HPLC. Individual peaks were desalted and analyzed by MALDI-TOF mass spectrometry; observed m/z are reported in Table 2 alongside their structural interpretations. (D) Diagram of cross-linked S. aureus peptidoglycan with arrows identifying the bonds that are cleaved by mutanolysin, lysostaphin, and EssH. mAU, milli-absorbance units; green and blue hexagons, N-acetylglucosamine and N-acetylmuramic acid, respectively.