The specificity of Ssp1 and Ssp2. (a) Peptidoglycan from E. coli strain D456 was incubated with Ssp1, Ssp2 or no enzyme (control) followed by digestion with the muramidase Cellosyl and analysis of the resulting muropeptides by HPLC. Both Ssp1 and Ssp2 cleaved non-cross-linked (tri, tetra and penta) and cross-linked (tetratetra and tetrapenta) muropeptides between D-glutamate and meso-diaminopimelic acid (m-A2pm), resulting in the disaccharide dipeptide (di) product. Ssp2 also produced a small amount of the disaccharide hexapeptide (hexa) product. (b) Proposed structures of the muropeptides separated in (a). The arrows indicate the cleavage sites of D-iGlu-m-A2pm endopeptidases in peptidoglycan. GlcNAc, N-acetylglucosamine; MurNAc(r), N-acetylmuramitol; m-A2pm, meso-diaminopimelic acid.