Mur-LH, the Broad-Spectrum Endolysin of Lactobacillus helveticus Temperate Bacteriophage phi-0303
2004
Deutsch, Stéphanie-Marie | Guézenec, Stéphane | Piot, Michel | Foster, Simon | Lortal, Sylvie | Science et Technologie du Lait et de l'Oeuf (STLO) ; Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST | Department of Molecular Biology and Biotechnology ; University of Sheffield [Sheffield]
-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycinC induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a libraryof -0303 in Escherichia coli DH5 . The lytic activity was detected by its expression, using whole cells of thesensitive 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 holingenes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, butit was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCRamplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a proteinof 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons withsequences 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 electrophoresiswith L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecularmass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls ofL. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated thatMur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, asit was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillussubtilis, Brevibacterium linens, and Enterococcus faecium.
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