HETEROGENEOUS ENDOLYSINS IN LISTERIA-MONOCYTOGENES BACTERIOPHAGES - ANEW CLASS OF ENZYMES AND EVIDENCE FOR CONSERVED HOLIN GENES WITHIN THE SIPHOVIRAL LYSIS CASSETTES

Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recomb inant cells were overlaid with a lawn of Listeria cells. The nucleotid e sequences of the cloned DNA fragments were determined and the indivi dual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) we re shown to have varying degrees of homology within their N-terminal o r C-terminal domains. Transcriptional analysis revealed them to be 'la te' genes with transcription beginning 15-20 min post-infection. The e nzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced r apid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell wal ls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enz ymes of this type. In contrast, PLY118 and PLY500 were shown to repres ent a new class of cell wall lytic enzymes which cleave between the L- alanine and D-glutamate residues of listerial peptidoglycan; these wer e designated as L-alanoyl-D-glutamate peptidases. These two enzymes sh are homology in the N-terminal domain which we propose determines hydr olytic specificity. Highly conserved holin (hol) gene sequences are pr esent upstream of ply118 and ply500. They encode proteins of structura l similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holi n genes with downstream lysin genes among the siphoviral lysis cassett es explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.