Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes

The genes encoding the host cell wall-lytic proteins were searched in the g enome DNA of phage PL-1 active against Lactobacillus casei ATCC 27092 by co mparing the amino acid sequences with those of others using a computer soft ware of the DDBJ data base. The gene regions found were cloned into E. coli by inserting PCR-amplified DNA fragments into the EcoRI site of pUC 19, an d the nucleotide sequences were determined. One of the ORFs (hol) consisted of 270 bp encoding 90 amino acids. The hol product (holin) possessed a put ative secretion signal, two putative transmembrane helices, and a highly ch arged C-terminus. Another ORF (lys) consisted of 1050 bp encoding an N-acet ylmuramoyl-L-alanine amidase of 350 amino acids. The gene lys was expressed in E, coli using pCALn expression vector, and the purified gene product hy drolysed the amide linkage in the peptidoglycans of L. casei. The amino aci d sequence of PL-1 amidase showed a high homology to those of Lactococcus l actis phage rlt and Lister in monocyto-genes phage A511. It was suggested t hat the N-terminal region was involved in enzyme activity and the C-termina l region in binding the enzyme to the cell wall substrate, respectively.