FIGURE 4.

Engineering a loss-of-function (muramidase/lysozyme) lysin. A, comparison of the lytic activities of full-length and catalytic domains of B. anthracis phage lysozymes, PlyBa04 and PlyB, against B. cereus ATCC 4243. The data for PlyB are derived from Fig. 1 of Porter et al. (47). Enzyme and bacterial concentrations and strains are identical. B, PlyBa04^CAT and PlyL^CAT have similar host ranges and synergize in killing B. cereus. Left panel, PlyBa04^CAT is a highly effective killer of B. cereus, B. megaterium, and B. subtilis (t½ = 50–100 s with 0.8 μm lysin), similar to the behavior of PlyL^CAT (and faster than the full-length protein in each case). Right panel, when used in combination, there is a clear synergistic effect between PlyL^CAT and PlyBa04^CAT against B. cereus. Total lysin concentration is the same in each case (0.4 μm). P/B = 0.2 μm PlyL^CAT + 0.2 μm PlyBa04^CAT). C, stereo overlay of PlyBa04 (cyan) and PlyB (gray) catalytic domains, with secondary structure elements as in C. The four mutation sites in PlyBa04^CAT are indicated. The two pairs of conserved acidic residues at the active site are shown as ball-and-stick (PlyBa04 numbering), as is a molecule of buffer (MES), which lines the center of the site and may be a substrate mimetic. A glycan-muropeptide fragment is modeled based on an overlay with the structure of pneumococcal phage lysin, Cpl-1 (Protein Data Bank code 1H09), in complex with a fragment of PG. E, comparison of lytic activities of PlyBa04 wild-type and mutant lysins against B. cereus in the context of full-length and catalytic domains. D, structure-based sequence alignment and secondary structure assignments of PlyBa04^CAT and PlyB^CAT, with active residues boxed. Mutations sites are numbered and labeled with *.