Mur-LH, the Broad-Spectrum Endolysin of Lactobacillus helveticus Temperate Bacteriophage phi-0303
Résumé
-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin
C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library
of -0303 in Escherichia coli DH5 . The lytic activity was detected by its expression, using whole cells of the
sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of
-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin
genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but
it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR
amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein
of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with
sequences in sequence databases showed homology with numerous endolysins of other bacteriophages.
Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis
with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular
mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of
L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that
Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as
it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus
subtilis, Brevibacterium linens, and Enterococcus faecium.