3 BACILLUS-CEREUS BACTERIOPHAGE ENDOLYSINS ARE UNRELATED BUT REVEAL HIGH HOMOLOGY TO CELL-WALL HYDROLASES FROM DIFFERENT BACILLI

The ply genes encoding the endolysin proteins from Bacillus cereus pha ges Bastille, TP21, and 12826 were identified, cloned, and sequenced, The endolysins could be overproduced in Escherichia coil (up to 20% of total cellular protein), and the recombinant proteins were purified b y a two step chromatographical procedure, All three enzymes induced ra pid and specific lysis of viable cells of several Bacillus species,,vi th highest activity on B, cereus and B, thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetyl muramoyl-L-alanine amidases (EC 3.5.1.28), No apparent holin genes were found adjacent to the ply genes, However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysi n. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply 12, 27.7 kDa) show remarkable heterogeneity, i.e,, their amino acid se quences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp , cell wall hydrolase (CmISP) and an autolysin of B, subtilis (CwlA), The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP, Ply12 shows homolo gy to the major autolysins from B, subtilis and E, coil, Comparison,vi th database sequences indicated a modular organization of the phage ly sis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relati onship of the phage enzymes and cell wall autolysins is based upon hor izontal gene transfer among different Bacillus phages and their hosts.