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

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 genome DNA of phage PL-1 active against Lactobacillus case i ATCC 27092 by comparing the amino acid sequences with those of others using a computer software of the DDBJ data base. The gene regions found were cloned into E. coli by inserting PCR-amplified DNA fragments into the Eco RI site of pUC19, and the nucleotide sequences were determined. One of the ORFs ( hol ) consisted of 270 bp encoding 90 amino acids. The hol product (holin) possessed a putative secretion signal, two putative transmembrane helices, and a highly charged C-terminus. Another ORF ( lys ) consisted of 1050 bp encoding an N -acetylmuramoyl-L-alanine amidase of 350 amino acids. The gene lys was expressed in E. coli using pCALn expression vector, and the purified gene product hydrolysed the amide linkage in the peptidoglycans of L. casei . The amino acid sequence of PL-1 amidase showed a high homology to those of Lactococcus lactis phage rlt and Listeria monocytogenes phage A511. It was suggested that the N-terminal region was involved in enzyme activity and the C-terminal region in binding the enzyme to the cell wall substrate, respectively. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2000 by Springer-Verlag/Wien
Subject
Legacy
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s007050070073
Publisher site
See Article on Publisher Site

Abstract

The genes encoding the host cell wall-lytic proteins were searched in the genome DNA of phage PL-1 active against Lactobacillus case i ATCC 27092 by comparing the amino acid sequences with those of others using a computer software of the DDBJ data base. The gene regions found were cloned into E. coli by inserting PCR-amplified DNA fragments into the Eco RI site of pUC19, and the nucleotide sequences were determined. One of the ORFs ( hol ) consisted of 270 bp encoding 90 amino acids. The hol product (holin) possessed a putative secretion signal, two putative transmembrane helices, and a highly charged C-terminus. Another ORF ( lys ) consisted of 1050 bp encoding an N -acetylmuramoyl-L-alanine amidase of 350 amino acids. The gene lys was expressed in E. coli using pCALn expression vector, and the purified gene product hydrolysed the amide linkage in the peptidoglycans of L. casei . The amino acid sequence of PL-1 amidase showed a high homology to those of Lactococcus lactis phage rlt and Listeria monocytogenes phage A511. It was suggested that the N-terminal region was involved in enzyme activity and the C-terminal region in binding the enzyme to the cell wall substrate, respectively.

Journal

Archives of VirologySpringer Journals

Published: Aug 1, 2000

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